scholarly journals RNA turnover in Trypanosoma brucei.

1987 ◽  
Vol 7 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
B Ehlers ◽  
J Czichos ◽  
P Overath
Keyword(s):  
Protein Synthesis ◽  
Trypanosoma Brucei ◽  
Half Life ◽  
De Novo ◽  
State Level ◽  
Surface Glycoprotein ◽  
Beta Tubulin ◽  
Fold Reduction ◽  
Tubulin Mrna

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

RNA turnover in Trypanosoma brucei

1987 ◽  
Vol 7 (3) ◽  
pp. 1242-1249
Author(s):  
B Ehlers ◽  
J Czichos ◽  
P Overath
Keyword(s):  
Protein Synthesis ◽  
Trypanosoma Brucei ◽  
Half Life ◽  
De Novo ◽  
State Level ◽  
Surface Glycoprotein ◽  
Beta Tubulin ◽  
Fold Reduction ◽  
Tubulin Mrna

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

scholarly journals In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

2006 ◽  
Vol 5 (8) ◽  
pp. 1276-1286 ◽  
Author(s):  
Sara D. Faulkner ◽  
Monika W. Oli ◽  
Rudo Kieft ◽  
Laura Cotlin ◽  
Justin Widener ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Trypanosoma Brucei ◽  
Density Lipoprotein ◽  
High Density ◽  
Surface Glycoprotein ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Expression Site ◽  
Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

Influence of ammonia concentration on [15N]ammonia uptake andde novosynthesis of different amino acids by mixed rumen microorganisms from the sheep rumenin vitro

1999 ◽  
Vol 1999 ◽  
pp. 212-212 ◽  
Author(s):  
C. Atasoglu ◽  
C.J. Newbold ◽  
R.J. Wallace
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
De Novo ◽  
Ammonia Concentration ◽  
Dependent Manner ◽  
Microbial Protein ◽  
Concentration Dependent Manner ◽  
Rumen Microorganisms ◽  
Ammonia Uptake

Ammonia is thought to be the main source of nitrogen for protein synthesis by the rumen microorganisms, but peptides and amino acids derived from protein degradation are also incorporated into microbial protein. Recent experiments carried out by Atasogluet al.(1998) demonstrated that preformed amino acids decrease the uptake of ammonia into microbial protein and microbial amino acids in a concentration-dependent manner. However, little is known about how rumen ammonia concentrations affect ammonia uptake into microbial protein. The present study was undertaken to determine the influence of rumen ammonia concentrations on ammonia incorporation andde novosynthesis of individual amino acids by the mixed rumen microorganismsin vitro.

scholarly journals Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N -Acetyltransferase from Trypanosoma brucei

2011 ◽  
Vol 10 (7) ◽  
pp. 985-997 ◽  
Author(s):  
Karina Mariño ◽  
M. Lucia Sampaio Güther ◽  
Amy K. Wernimont ◽  
Wei Qiu ◽  
Raymond Hui ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Trypanosoma Brucei ◽  
Protein Glycosylation ◽  
Surface Glycoprotein ◽  
Surface Coat ◽  
Content Type ◽  
Phosphate Analysis ◽  
Novel Drug

ABSTRACT A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N -acetyltransferase ( TbGNA1 ; EC 2.3.1.4) was cloned and expressed in Escherichia coli . The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly- N -acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.

scholarly journals Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

1994 ◽  
Vol 5 (7) ◽  
pp. 819-828 ◽  
Author(s):  
Y Wang ◽  
G M Fuller
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
De Novo ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Synthesis Inhibitors ◽  
Different Cell Types ◽  
Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

scholarly journals Endothelial cells secrete a factor that promotes fibroblast contraction of hydrated collagen gels.

1990 ◽  
Vol 110 (2) ◽  
pp. 519-528 ◽  
Author(s):  
C Guidry ◽  
S Hohn ◽  
M Hook
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Biochemical Characterization ◽  
De Novo ◽  
Collagen Gels ◽  
Serum Factors ◽  
Inhibitor Of Protein Synthesis

Bovine aortic endothelial cells (BAEC), grown in vitro, are shown to synthesize and secrete factor(s) that stimulate fibroblasts to contract collagen matrices. The amount of contraction-promoting activity in the conditioned media is dependent on conditioning time and the number of cells in the culture. Production of the contraction-promoting activity continues at a high stable level for at least 5 d in serum-free medium but is abolished when the cells are exposed to an inhibitor of protein synthesis. The mechanism of action of the contraction factor(s) derived from endothelial cells was compared with that of unidentified serum factors. The endothelial cell-secreted factor(s) depends on active protein synthesis by the target cell but does not need to be present during the contraction process. The serum factors on the other hand promote collagen contraction in the absence of de novo protein synthesis but need to be continuously present. Preliminary biochemical characterization of the contraction-promoting factors produced by endothelial cells revealed properties similar to those of previously identified growth factors. However, the BAEC-secreted factor was found to be distinct from a previously identified contraction-promoting transforming growth factor beta.

In Vitro Induction of Tyrosine Aminotransferase in Liver Slices by Hydrocortisone

1971 ◽  
Vol 49 (12) ◽  
pp. 1357-1361 ◽  
Author(s):  
J. P. Liberti ◽  
C. H. DuVall ◽  
D. M. Wood
Keyword(s):  
Protein Synthesis ◽  
Half Life ◽  
Liver Slice ◽  
Tyrosine Aminotransferase ◽  
Maximal Response ◽  
Liver Slices ◽  
Hypophysectomized Rats ◽  
Liver Homogenates ◽  
In Vitro Induction

A liver slice system has been developed which actively incorporates labeled precursors into RNA and protein for as long as 10 h of incubation. The addition of glucocorticoid induces tyrosine aminotransferase (TA) in liver slices of hypophysectomized rats but not in liver homogenates of hypophysectomized rats or in slices derived from normal rats. TA activity is increased two- to sixfold during 6 h of incubation, the maximal response being observed with 10−6 M steroid. Addition of cydoheximide, at a concentration which depresses protein synthesis 95%, blocks the accumulation of TA. The half-life [Formula: see text] of induced TA is approximately 2–3 h and is similar to that of the basal, noninduced system. These data indicate that glucocorticoid acts only to increase the rate of TA synthesis without affecting its rate of degradation.

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