The Effect of Neuraminidase (3:2:1:18) on the Aggregation of Cells Dissociated from Embryonic Chick Muscle Tissue

1970 ◽  
Vol 6 (3) ◽  
pp. 751-766
Author(s):  
R. B. KEMP
Keyword(s):  
Cell Surface ◽  
Muscle Tissue ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Sialic Acids ◽  
Embryonic Chick ◽  
Experimental Conditions ◽  
Cell Counts

Embryonic chick muscle cells were used to investigate the effect of removing cell-surface sialic acids on cell aggregation in vitro. Single cell suspensions were prepared by dissociating skeletal muscle tissue of 9-day-old chick embryos with either crystalline or crude trypsin. Cell aggregation was quantitatively estimated by turbidimetric and gyratory shaker methods. Cells dissociated with crude trypsin and suspended in Hanks's balanced salts solution (BSS) containing 25u./ml neuraminidase (NANase) only aggregated for 2h when rotated in an absorptiometer. The inhibitory effect of the enzyme was more pronounced with increasing concentration up to 25u./ml. Cells dissociated with crystalline trypsin and treated with 100u./ml NANase immediately exhibited a reduced aggregative competence when gyrated in Eagle's minimum essential medium (MEM) containing 25u./ml NANase, compared with the controls which were not exposed to NANase. The aggregation rate of muscle cells pretreated with 100u./ml NANase and suspended in Eagle's MEM was similar to that of the untreated controls. Cell counts showed that under all three experimental conditions cells were not added to aggregates after the 12-h stage. Aggregates formed in Eagle's MEM (the controls) joined together to form larger aggregates after 12 h, but those rotating in the presence of NANase did not display this property. Lissamine green viability tests showed that cells remained alive throughout the 24-h period in the presence of NANase. Determinations of oxygen uptake, protein synthesis and mitotic index confirmed that general cellular viability was not affected by NANase. Fluorescent-labelled NANase was not taken up by the cells. Treatment of crystalline trypsin-dissociated muscle cells with 100u./ml NANase for 30 min at 37°C significantly reduced their negative electrophoretic mobility. This diminution closely corresponded to the removal of cell-surface sialic acids, as measured by colorimetric tests. Interpretation of the results in the light of current theories of cell adhesion failed to give support to the concept of adhesion by physical forces. The mechanism by which cellular deformability could influence cellular adhesiveness is modified in the knowledge of the present results.

Aggregative Behaviour of Embryonic Chick Cells in the Presence of Anti-Bodies Directed Against Actomyosins

1971 ◽  
Vol 9 (1) ◽  
pp. 103-122
Author(s):  
R. B. KEMP ◽  
B. M. JONES ◽  
U. GRÖSCHEL-STEWART
Keyword(s):  
Smooth Muscle ◽  
Cell Surface ◽  
Striated Muscle ◽  
Calf Serum ◽  
Fluorescein Isothiocyanate ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Human Umbilical Cord ◽  
Gyratory Shaker

Skeletal muscle and liver tissue from 9-day-old chick embryos were dissociated into separate cells using 0.25 % (w/v) crude trypsin. The effect of rabbit anti-actomyosin sera on the aggregation of these cells was estimated by the gyratory shaker and turbidimetric methods. Studies were also undertaken on the ability of fluorescein isothiocyanate-labelled rabbit anti-uterine actomyosin serum (FITC-labelled anti-UAM) to stain the cell surface and on the type specificity and species specificity of rabbit anti-chicken actomyosin sera. Antisera against chicken gizzard smooth-muscle actomyosin (anti-GAM) and against chicken pectoralis striated muscle actomyosin (anti-PAM) both gave single precipitin bands with their respective actomyosins on diffusion through agar. The antisera neither reacted with their heterologous actomyosin nor with gizzard tropomyosin; they were type-specific. Serial sections of human cervix were stained in a similar pattern with both anti-UAM and anti-GAM, showing that anti-smooth muscle actomyosin sera were not species-specific. The fibrocytes of the human umbilical cord and human platelets were stained by FITC-labelled anti-UAM serum but not by labelled anti-human PAM. The aggregation of muscle and liver cells over a 24-h period in the presence of antisera against human or chicken PAM was not significantly different from the controls incubated on a gyratory shaker in Eagle's minimum essential medium (MEM) containing 10% (v/v) rabbit non-immunized serum (NIS) or calf serum. However, anti-UAM and anti-GAM inhibited both the rate of aggregation of liver and muscle cells and the size of aggregates attained in 24 h. This effect could not be simulated with specific rabbit antisera against human plasma proteins. The globulin-enriched fraction of anti-GAM markedly inhibited the aggregation of liver and muscle cells in a range of concentrations between 50 and 500 µg per 2 x 106 cells/ml Eagle's MEM. In contrast, the aggregation of cells incubated with globulin-enriched anti-PAM was similar to the controls. The addition of anti-GAM globulins at 1 or 2 h to muscle cells rotated by the turbidimetric method reduced the aggregative competence of the cells over the remainder of a 4-h period. The possibility that the inhibitory effect of anti-UAM and anti-GAM on cell aggregation is due to impurities in the antisera or to a general reaction with cell surface ATPases is discussed but, in the light of evidence, rejected in favour of a reaction between the antisera and an actomyosin of the smooth-muscle type at the cell surface.

Effect of the Removal of Cell Surface Sialic Acids on Cell Aggregation in vitro

Nature ◽  
1968 ◽  
Vol 218 (5148) ◽  
pp. 1255-1256 ◽  
Author(s):  
R. B. KEMP
Keyword(s):  
Cell Surface ◽  
Cell Aggregation ◽  
Sialic Acids

Abolition by Myosin and Heavy Meromyosin of the Inhibitory Effect of Smooth-Muscle Actomyosin Antibodies on Cell Aggregation In Vitro

1973 ◽  
Vol 12 (2) ◽  
pp. 631-639
Author(s):  
R. B. KEMP ◽  
B. M. JONES ◽  
U. GRÖSCHEL-STEWART
Keyword(s):  
Smooth Muscle ◽  
Striated Muscle ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Regulatory Function ◽  
Rabbit Serum ◽  
Heavy Meromyosin ◽  
Γ Globulins ◽  
Dissociated Cells

The ability of anti-chicken smooth-muscle actomyosin γ-globulins (anti-GAM) to inhibit the aggregation of dissociated cells from the skeletal muscle and liver of chick embryos was abolished by pretreatment of the anti-GAM with either myosin or heavy meromyosin (HMM). When the same cells were treated with HMM at a concentration of 1 mg per 2 x 106 cells/ml Eagle's MEM they aggregated as readily as untreated cells. The negative electrophoretic mobility of the embryonic chick fibroblastic cells was significantly reduced by the globulin fraction of anti-GAM but not of HMM-treated anti-GAM or non-immunized rabbit serum. Anti-chicken striated muscle actomyosin γ-globulins slightly reduced negative mobility but HMM had no effect. The experiments show that the inhibitory effect on cell aggregation of anti-GAM preparations is produced by the anti-myosin antibodies. They also provide support for the theory that a surface-localized myosin-like protein has a regulatory function in cell adhesion.

scholarly journals Angiotensin II inhibits NaCl absorption in the rat medullary thick ascending limb

2004 ◽  
Vol 287 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
Nicolas Lerolle ◽  
Soline Bourgeois ◽  
Françoise Leviel ◽  
Gaëtan Lebrun ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Plasma Membranes ◽  
Inhibitory Effect ◽  
Thick Ascending Limb ◽  
Sprague Dawley ◽  
Experimental Conditions ◽  
Nacl Absorption ◽  
Medullary Thick Ascending Limb

NaCl reabsorption in the medullary thick ascending limb of Henle (MTALH) contributes to NaCl balance and is also responsible for the creation of medullary interstitial hypertonicity. Despite the presence of angiotensin II subtype 1 (AT1) receptors in both the luminal and the basolateral plasma membranes of MTALH cells, no information is available on the effect of angiotensin II on NaCl reabsorption in MTALH and, furthermore, on angiotensin II-dependent medullary interstitial osmolality. MTALHs from male Sprague-Dawley rats were isolated and microperfused in vitro; transepithelial net chloride absorption ( JCl) as well as transepithelial voltage ( Vte) were measured. Luminal or peritubular 10−11 and 10−10 M angiotensin II had no effect on JCl or Vte. However, 10−8 M luminal or peritubular angiotensin II reversibly decreased both JCl and Vte. The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10−6 M). By contrast, PD-23319, an AT2-receptor antagonist, did not alter the inhibitory effect of 10−8 M angiotensin II. Finally, no additive effect of luminal and peritubular angiotensin II was observed. We conclude that both luminal and peritubular angiotensin II inhibit NaCl absorption in the MTALH via AT1 receptors. Because of intrarenal angiotensin II synthesis, angiotensin II concentration in medullary tubular and interstitial fluids may be similar in vivo to the concentration that displays an inhibitory effect on NaCl reabsorption under the present experimental conditions.

A novel mechanism of olanzapine-induced lipid accumulation

2011 ◽  
Vol 26 (S2) ◽  
pp. 906-906 ◽  
Author(s):  
S. Dzitoyeva ◽  
H. Chen ◽  
R. Manev ◽  
H. Manev
Keyword(s):  
Lipid Content ◽  
Direct Action ◽  
Inhibitory Effect ◽  
Experimental Conditions ◽  
Cell Content ◽  
Metabolic Alterations ◽  
Low Concentrations ◽  
Mrna Content ◽  
Quantitative Western Blot

IntroductionSecond generation antipsychotic drugs (SGADs) including olanzapine trigger adverse metabolic alterations possibly by a direct action on adipocytes.Objectives and aimsThe system of the inflammatory 5-lipoxygenase (5-LOX) and its activating protein (FLAP) have been implicated in lipid dysfunction in obesity. We investigated whether this system could participate in the adipogenic action of olanzapine.MethodsExperiments were performed in 3T3-L1 adipocytes in vitro. Cells were treated with olanzapine and a FLAP inhibitor MK-886. Their lipid content, 5-LOX and FLAP mRNA content, and FLAP protein content were measured.ResultsOlanzapine treatment did not affect the cell content of 5-LOX mRNA; however, it decreased FLAP mRNA content at day five but not 24 hours after olanzapine addition. The inhibitory effect of olanzapine on FLAP expression was confirmed by quantitative Western blot assays. In the absence of a FLAP inhibitor, low concentrations of olanzapine (0.5 and 5 μM) increased lipid content only by about 13% (compared to about a 56% increase induced by 50 μM olanzapine) whereas in the presence of MK-886 these concentrations of olanzapine produced lipid increases comparable to the increase caused by 50 μM. In these experimental conditions, MK-886 alone did not alter the cell content of lipids.Conclusions5-LOX system may be involved in lipid dysfunction not only in conditions of obesity but possibly in SGAD-related metabolic alterations. The known polymorphism in the genes of the human 5-LOX system could play a role in setting a variable individual susceptibility to the metabolic side effects of SGADs.

CONTRIBUTION OF MATHEMATICAL MODELLING TO ELUCIDATION OF AIDS PATHOGENESIS

1993 ◽  
Vol 01 (04) ◽  
pp. 349-362 ◽  
Author(s):  
TOMÁŠ HRABA ◽  
JAROSLAV DOLEŽAL
Keyword(s):  
Mathematical Model ◽  
Hiv Infection ◽  
Mathematical Modelling ◽  
Immune Reaction ◽  
Inhibitory Effect ◽  
Immunological Tolerance ◽  
Lymphocyte Depletion ◽  
Cell Counts ◽  
Hiv Research

Although HIV is cytopathogenic for CD4+ lymphocytes in vitro, the depletion of these cells in HIV-infected individuals seems to be caused by some other mechanism. It is probably a direct destructive or inhibitory effect of the viral products or an (auto)immune reaction elicited by them, eventually directed at them. Mathematical model of immunological tolerance was used for simulation of CD4+ lymphocyte depletion dynamics in HIV infection under the assumption that it is caused by HIV products. This model was not able to simulate the observed three-stage dynamics of CD4+ cell counts, i.e., drop of these counts around seroconversion, followed by a slow decrease over a prolonged period and an accelerated decrease before the development of AIDS. When an immune reaction limiting HIV reproduction was included in the model, all these three phases of CD4+ lymphocyte depletion could be simulated. In the paper, different modifications and extensions of the model are reviewed, compared with available clinical data, and their relevance for HIV research is discussed.

scholarly journals A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION

1974 ◽  
Vol 60 (3) ◽  
pp. 641-652 ◽  
Author(s):  
Joris J. Deman ◽  
Erik A. Bruyneel ◽  
Marc M. Mareel
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Calcium Ions ◽  
Hela Cells ◽  
Previous Treatment ◽  
Cell Aggregation ◽  
Sialic Acids ◽  
Magnesium Ions ◽  
Trypsin Treatment ◽  
Calcium Effect

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.

scholarly journals THE FINE STRUCTURE OF EMBRYONIC CHICK SKELETAL MUSCLE CELLS DIFFERENTIATED IN VITRO

1967 ◽  
Vol 35 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Y. Shimada ◽  
D. A. Fischman ◽  
A. A. Moscona
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Fine Structure ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Developing Muscle

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

scholarly journals Role of CD40 Ligand in Mycobacterium avium Infection

1999 ◽  
Vol 67 (7) ◽  
pp. 3558-3565 ◽  
Author(s):  
Tomoko Hayashi ◽  
Savita P. Rao ◽  
Pascal R. Meylan ◽  
Richard S. Kornbluth ◽  
Antonino Catanzaro
Keyword(s):  
Mycobacterium Avium ◽  
Human Monocyte ◽  
Opportunistic Pathogen ◽  
Cd40 Ligand ◽  
Inhibitory Effect ◽  
Interleukin 12 ◽  
Intracellular Growth ◽  
Cell Counts ◽  
Avium Infection

ABSTRACT Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4+ cell counts of <50 cells/μl, suggesting a possible involvement of a T cell-macrophage interaction for the elimination of M. avium. To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. aviuminfection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth ofM. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% ± 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 μg/ml) inhibited intracellular growth of M. avium by 76.9% ± 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium-infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium-infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.

Studies on the Mechanism Underlying the Inhibition by Puromycin of Cell Aggregation In Vitro

1970 ◽  
Vol 7 (2) ◽  
pp. 557-573
Author(s):  
M. J. DUNN ◽  
E. OWEN ◽  
R. B. KEMP
Keyword(s):  
Carbon Dioxide ◽  
Protein Synthesis ◽  
Cellular Metabolism ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Inhibitory Effect ◽  
Dissociated Cells ◽  
Gyratory Shaker ◽  
Reduced Rate

Cells dissociated with 0.25% crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10µg/ml into a suspension of cells in Eagle's MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α-14C]leucine into proteins, was arrested by 91.7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40% within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further 14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle's MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0.6% between the glycogen content of treated cells and controls. At 24 h 11.3% less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2.59 to 1.45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0.62 at 4 h and had further declined to 0.48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

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