Mechanisms involved in the loss or antibody-mediated adherence of macrophages to lung-stage schistosomula of Schistosoma mansoni in vitro

Parasitology ◽  
1993 ◽  
Vol 106 (5) ◽  
pp. 463-469 ◽  
Author(s):  
B. W. L. Lawson ◽  
Q. D. Bickle ◽  
M. G. Taylor
Keyword(s):  
Protein Synthesis ◽  
Immune Evasion ◽  
Actinomycin D ◽  
Cell Loss ◽  
Immune Serum ◽  
Adherent Cells ◽  
Cell Adherence ◽  
Rapid Loss ◽  
Antigen Loss

SUMMARYSera from rabbits vaccinated with irradiated cercariae mediated cell (P388D1 or mouse peritoneal macrophage) adherence to lung-stage schistosomula (LS) but such antibody-mediated cell adherence was short-lived in contrast to cell adherence to mechanically transformed schistosomula (MS). Thus LS lost 50% of their adherent cells within 3–6 h in culture and up to 90% by 24 h, whereas adherence to MS was undiminished during this time. Rapid loss of adherent cells was unique to schistosomula that had developed to the lung stage because schistosomula recovered from the skin up to 3 days post-infection did not exhibit the rapid cell loss shown by 3-day LS. To determine whether cell loss was caused by loss of surface antigenicity during culture LS were cultured on their own for up to 24 h and at various intervals samples of schistosomula were tested for antigenicity by addition of immune serum and cells. Levels of adherence to both MS and LS were maintained throughout the incubation period. When antibody-opsonized schistosomula were washed and indicator cells added at progressive intervals, persistence of adherence was again demonstrated, showing that antibody binding to LS had not promoted surface antigen loss or degradation of bound antibody. It was then shown, by adding fresh macrophages to cultures up to 24 h old that LS which had lost their adherent cells nevertheless retained bound antibody, and comparison of adherence of ‘used’ and ‘fresh’ cells to MS and LS showed that the cytoadherence properties of macrophages were not significantly reduced during their culture with LS from which cells had been lost. Cell loss was shown to be dependent upon protein synthesis by LS since cell loss was significantly reduced when the schistosomula were pre-incubated in puromycin or actinomycin D. Transient cell adherence to LS may comprise an important immune evasion stratagem by schistosomula.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

Activation of Protein Synthesis in Aging Potato Tuber Slices

1971 ◽  
Vol 26 (10) ◽  
pp. 1064-1067 ◽  
Author(s):  
Günter Kahl
Keyword(s):  
Protein Synthesis ◽  
Potato Tuber ◽  
Actinomycin D ◽  
Leucine Incorporation ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Fresh Potato ◽  
Polyuridylic Acid ◽  
Storage Organs

Whereas ribosome preparations of freshly sliced potato disks do not show appreciable activity in an in-vitro amino acid incorporation system, aging of the tissue leads to a greatly enhanced incorporation activity which reaches its maximum 24 hours after slicing. If ribosomes from freshly excised disks are provided with polyuridylic acid, their activity in the incorporation of phenylalanine is increased about 8 fold.Moreover, an RNA-fraction can be dissociated by EDTA from ribosomes of aged potato tuber slices, which sediments at 15 —18S, has a base composition different from that of 16S — rRNA, 5S-and 4S —RNA, and is not present on ribosomes of fresh slices. Its appearance is inhibited by actinomycin D and therefore most probably dependent on transcription. This compound, purified from sucrose gradients, enhances in vitro leucine incorporation into peptide material by ribosomes of fresh potato slices.The possibility is discussed that this fraction-among other factors-is responsible for the enhanced protein synthesis after slicing plant storage organs, and is indicative of a general derepression phenomenon in these tissues.

Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock

1984 ◽  
Vol 307 (1132) ◽  
pp. 301-307 ◽  
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Mammalian Cells ◽  
Actinomycin D ◽  
Future Studies ◽  
Cos Cells ◽  
Single Protein ◽  
Cellular Components ◽  
Multiple Interactions

We transformed mouse L cells with a cloned Drosophila hsp70 gene and obtained cells with either heat-inducible or constitutively expressed copies of the gene. The distribution of hsp70 in these cells was examined by indirect immunofluorescence with monoclonal antibodies specific to the Drosophila protein. In constitutive cells, hsp70 was present in both cytoplasm and nucleus. After heat shock, the nuclear hsp70 was transiently concentrated in nucleoli, from which it had previously been excluded; the cytoplasmic hsp70 moved to a perinuclear location, a result consistent with it being associated with intermediate filaments of the cytoskeleton. The nucleolar migration took several hours and was partly inhibited by actinomycin D, but was independent of protein synthesis; it may reflect binding to newly-synthesized rRNA. Drosophila hsp70 was also expressed from replicating plasmids in monkey COS cells, and was found to be concentrated in nuclei even at low temperature. Migration to nucleoli occurred after heat shock. These results indicate that a single protein can have multiple interactions with cellular components, and form the basis for future studies of these interactions by in vitro mutagenesis and expression of the hsp70 gene.

scholarly journals Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2542-2547 ◽  
Author(s):  
Y Yamaguchi ◽  
T Suda ◽  
S Ohta ◽  
K Tominaga ◽  
Y Miura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Dna Fragmentation ◽  
Culture System ◽  
Actinomycin D ◽  
Agar Gel ◽  
Interleukin 5 ◽  
Liquid Culture System ◽  
Rna And Protein Synthesis ◽  
Agar Gel Electrophoresis

Abstract We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in an in vitro liquid culture system. Mature eosinophils did not proliferate but their survival was maintained in the presence of IL-5. Using this culture system, we investigated the mechanism of IL-5-mediated survival. In the absence of human IL-5 (hIL-5) mature eosinophils succumbed after 4 days, while in the presence of hIL-5 they survived up to 10 days. When DNA extracts of cultured eosinophils were analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hIL-5, while no significant DNA fragmentation was observed in the culture with hIL-5 for 48 hours. The DNA fragmentation appeared as early as 6 to 12 hours after hIL-5 deprivation. Concomitantly, IL-5 stimulated total RNA and protein synthesis, but did not induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protection of apoptosis by hIL-5, some new RNA and protein synthesis appeared to be required in this phenomena. These findings indicate that IL-5 maintains survival of mature eosinophils with induction of new RNA and protein synthesis, thus leading to the inhibition of apoptosis.

The role of serum in leucocyte adherence to the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea)

Parasitology ◽  
1986 ◽  
Vol 92 (2) ◽  
pp. 413-424 ◽  
Author(s):  
P. Hoole ◽  
C. Arme
Keyword(s):  
Rutilus Rutilus ◽  
Immune Serum ◽  
Adherent Cells ◽  
Percoll Gradient ◽  
Serum Dilution ◽  
Ligula Intestinalis ◽  
Discontinuous Percoll Gradient ◽  
Leucocyte Adherence

SUMMARYThe role of serum in the adherence of roach (Rutilus rutilus) leucocytes to the plerocercoid of Ligula intestinalis has been investigated in vitro. Roach plerocercoids, either untreated, cultured or killed (fixation in 0·5 % buffered glutaraldehyde or heat at 50°C for 45 mm), were exposed to serum and 4×106 leucocytes obtained from the pronephros of non-infected roach using a discontinuous Percoll gradient. At normal roach serum (NRoS) dilutions of 1:5000 to 1:100, leucocyte adherence was observed on all living parasites but was negligible in killed parasites or in complement-depleted NRoS assays. The number of adherent cells increased with increasing NRoS concentration. Leucocytes bound more avidly in the presence of heat-inactivated immune serum (hi IRoS); at a serum dilution of 1:5000 the percentage of the parasite surface covered by leucocytes was approximately 3 times greater in hi IRoS than in NRoS. Ultrastructural observations on parasites revealed that plerocercoids exposed to cells and hi IRoS had, in some areas, abnormal inclusions in their tegument and lacked a microthrix border and distal cytoplasm. In addition, the efflux of radioactivity from [14C] cycloleucine-labelled worms was greater in parasites incubated in the presence of hi IRos.

THE DEVELOPMENT OF AN IN VITRO SYSTEM FOR ESTIMATING OXYTOCINASE IN THE RABBIT HYPOTHALAMUS AND THE EFFECTS OF SOME PROTEIN SYNTHESIS INHIBITORS AND OESTRADIOL-17β ON ENZYME ACTIVITY

1974 ◽  
Vol 75 (3) ◽  
pp. 443-451 ◽  
Author(s):  
Dona A. Frith ◽  
K. C. Hooper
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Steroid Hormones ◽  
Actinomycin D ◽  
Protein Synthesis Inhibitors ◽  
In Vitro System

ABSTRACT An in vitro system for investigating the effects of steroid hormones and protein synthesis inhibitors on hypothalamic peptidases inactivating oxytocin has been developed. In the presence of oestradiol-17β enzyme activity was increased in the in vitro system whilst this increase was blocked completely by cycloheximide and partially blocked by actinomycin-D. It is apparent therefore that oestradiol-17β acts directly on the hypothalamus stimulating oxytocinase activity.

scholarly journals The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats

1981 ◽  
Vol 196 (2) ◽  
pp. 383-390 ◽  
Author(s):  
M J Wakelam ◽  
D G Walker
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Actinomycin D ◽  
Neonatal Rats ◽  
Dibutyryl Cyclic Amp ◽  
Subsequent Effect ◽  
Old Rats ◽  
Effect Of Glucose

1. The specificity of the effect of glucose on the induction of glucokinase activity that occurs when hepatocytes freshly isolated from 13-day-old rats are incubated in Medium 199 together with insulin [Wakelam & Walker (1980) FEBS Lett. 111, 115-119] was examined. A pattern that is different from other known effects of glucose is found, and metabolism of this compound is not necessarily to account for this particular effect. 2. The effects of a raised glucose concentration and of insulin on the induction can be separated. The hexose initiates the process in the absence of insulin in a manner that is sensitive to actinomycin D but not to cycloheximide. The subsequent effect of insulin is dependent on the prior effect of glucose or other positive analogue, does not require the presence of glucose and is inhibited by cycloheximide but not by actinomycin D. 3. Induction of glucokinase in vitro in hepatocytes from neonatal animals is inhibited by adrenaline, glucagon and dibutyryl cyclic AMP, but not by vasopressin or angiotensin II. The inhibition by cyclic AMP is on the stage requiring insulin and is comparatively specific, because total protein synthesis is not apparently diminished. 4. The implications of these results are discussed with reference to possible mechanisms of induction and to the situation in vivo.

Lectin Stimulation of Tissue Thromboplastin Activity in Human Monocytes in vitro

1979 ◽  
Vol 42 (05) ◽  
pp. 1574-1579 ◽  
Author(s):  
T Lyberg ◽  
H Prydz
Keyword(s):  
Protein Synthesis ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Actinomycin D ◽  
De Novo ◽  
Human Monocytes ◽  
Tissue Thromboplastin ◽  
De Novo Protein Synthesis ◽  
Stimulation Of

SummaryLectins (phytohaemagglutinin, concanavalin A and wheat germ agglutinin) trigger an increase in tissue thromboplastin activity of human monocytes in vitro. The presence of serum was not necessary and did not enhance the activity. The increase was inhibited by cycloheximide and actinomycin D, suggesting that de novo protein synthesis is involved.

Adaptation of rabbit cortical collecting duct to in vitro acid incubation

1992 ◽  
Vol 263 (4) ◽  
pp. F749-F756 ◽  
Author(s):  
K. Yasoshima ◽  
L. M. Satlin ◽  
G. J. Schwartz
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Collecting Duct ◽  
Cytochalasin D ◽  
Reversible Inhibitor ◽  
Cortical Collecting Duct ◽  
Minimum Duration ◽  
Collecting Ducts ◽  
Inhibitor Of Protein Synthesis

Cortical collecting ducts (CCDs) isolated from acid-loaded rabbits and perfused in vitro absorb HCO3-, whereas CCDs from normal animals secrete HCO3-. We have previously shown that CCDs incubated in vitro for 3 h at pH 6.9 show a reduction in net (baseline and stimulated) HCO3- secretion. In this study we ascertained the minimum duration of an acidic stimulus necessary to induce adaptive changes in stimulated HCO3- secretion (determined in the absence of basolateral Cl-) and the roles of protein synthesis and cytoskeletal function in this process. CCDs incubated in acid (pH 6.8, HCO3- 6 mM) for 1 h followed by incubation at pH 7.4 (HCO3- 25 mM) for 2 h showed a 41% reduction in stimulated HCO3- secretion (P < 0.001), similar to that observed after 3 h of incubation at pH 6.8. However, this incubation protocol failed to enhance stimulated HCO3- absorption (determined in the absence of luminal Cl-). Addition of 10 microM anisomycin, a reversible inhibitor of protein synthesis, throughout the entire period of incubation (1 h at pH 6.8 plus 2 h at pH 7.4) blocked adaptive reduction in HCO3- secretion, as did exposure to anisomycin only during the initial 1 h of acid incubation. In contrast, anisomycin application during the 2-h incubation at pH 7.4 failed to block this adaptation of HCO3- secretion. Application of 4 microM actinomycin D, an inhibitor of DNA transcription, during the acid incubation also prevented the adaptive response, as did application during the total or during the 2-h pH 7.4 incubation period of 0.2 microM cytochalasin D, an inhibitor of actin filament function.(ABSTRACT TRUNCATED AT 250 WORDS)

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