scholarly journals Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.

1993 ◽  
Vol 13 (2) ◽  
pp. 1146-1154 ◽  
Author(s):  
F Bringaud ◽  
T Baltz
Keyword(s):  
Trypanosoma Brucei ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Protein S ◽  
Life Cycle Stage ◽  
Good Target ◽  
Hexose Transporters ◽  
Moderate Sensitivity ◽  
Mrna Analysis

A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2.

Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei

1993 ◽  
Vol 13 (2) ◽  
pp. 1146-1154
Author(s):  
F Bringaud ◽  
T Baltz
Keyword(s):  
Trypanosoma Brucei ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Protein S ◽  
Life Cycle Stage ◽  
Good Target ◽  
Hexose Transporters ◽  
Moderate Sensitivity ◽  
Mrna Analysis

A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2.

scholarly journals Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

1989 ◽  
Vol 9 (10) ◽  
pp. 4187-4195 ◽  
Author(s):  
J C Vera ◽  
O M Rosen
Keyword(s):  
Glucose Uptake ◽  
Rat Brain ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Rat Brain And Liver ◽  
Insulin Responsiveness

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.

scholarly journals Opposing Effects of Polyadenylation on the Stability of Edited and Unedited Mitochondrial RNAs in Trypanosoma brucei

2005 ◽  
Vol 25 (5) ◽  
pp. 1634-1644 ◽  
Author(s):  
Chia-Ying Kao ◽  
Laurie K. Read
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Stability ◽  
Protein S ◽  
Rna Degradation ◽  
Nucleotide Composition ◽  
Rna Turnover ◽  
Degradation Reactions ◽  
The Stability ◽  
Opposing Effects

ABSTRACT Mitochondrial RNAs in Trypanosoma brucei undergo posttranscriptional RNA editing and polyadenylation. We previously showed that polyadenylation stimulates turnover of unedited RNAs. Here, we investigated the role of polyadenylation in decay of edited RPS12 RNA. In in vitro turnover assays, nonadenylated fully edited RNA degrades significantly faster than its unedited counterpart. Rapid turnover of nonadenylated RNA is facilitated by editing at just six editing sites. Surprisingly, in direct contrast to unedited RNA, turnover of fully edited RNA is dramatically slowed by addition of a poly(A)20 tail. The same minimal edited sequence that stimulates decay of nonadenylated RNA is sufficient to switch the poly(A) tail from a destabilizing to a stabilizing element. Both nucleotide composition and length of the 3′ extension are important for stabilization of edited RNA. Titration of poly(A) into RNA degradation reactions has no effect on turnover of polyadenylated edited RNA. These results suggest the presence of a protective protein(s) that simultaneously recognizes the poly(A) tail and small edited element and which blocks the action of a 3′-5′ exonuclease. This study provides the first evidence for opposing effects of polyadenylation on RNA stability within a single organelle and suggests a novel and unique regulation of RNA turnover in this system.

scholarly journals Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

2021 ◽  
Author(s):  
Federico Rojas ◽  
Mathieu Cayla ◽  
Keith Matthews
Keyword(s):  
Quorum Sensing ◽  
Basement Membrane ◽  
Trypanosoma Brucei ◽  
Life Cycle Stage ◽  
Mammalian Host ◽  
Connective Tissues ◽  
Basement Membrane Extract ◽  
Differentiation Marker

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process.  For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo , we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms . BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro , procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity

1989 ◽  
Vol 9 (10) ◽  
pp. 4187-4195
Author(s):  
J C Vera ◽  
O M Rosen
Keyword(s):  
Glucose Uptake ◽  
Rat Brain ◽  
Xenopus Oocytes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Rat Brain And Liver ◽  
Insulin Responsiveness

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.

scholarly journals Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

2021 ◽  
Vol 15 (4) ◽  
pp. e0009284
Author(s):  
Federico Rojas ◽  
Mathieu Cayla ◽  
Keith R. Matthews
Keyword(s):  
Quorum Sensing ◽  
Basement Membrane ◽  
Trypanosoma Brucei ◽  
Life Cycle Stage ◽  
Mammalian Host ◽  
Connective Tissues ◽  
Basement Membrane Extract ◽  
Differentiation Marker

The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

Alkaloid subfractions of Buxus sempervirens L. leaves with strong in vitro activity against Trypanosoma brucei rhodesiense

Planta Medica ◽  
2014 ◽  
Vol 80 (16) ◽  
Author(s):  
JB Althaus ◽  
G Jerz ◽  
P Winterhalter ◽  
M Kaiser ◽  
R Brun ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Trypanosoma Brucei Rhodesiense ◽  
Buxus Sempervirens

Activated Protein C Increases Fibrin Clot Lysis by Neutralization of Plasminogen Activator Inhibitor No Evidence for a Cofactor Role of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 328-333 ◽  
Author(s):  
N J de Fouw ◽  
Y F de Jong ◽  
F Haverkate ◽  
R M Bertina
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Blood Platelets ◽  
Plasminogen Activator Inhibitor ◽  
Protein S ◽  
Tissue Type ◽  
Clot Lysis ◽  
Activator Inhibitor ◽  
Pai 1

summaryThe effect of purified human activated protein G (APC) on fibrinolysis was studied using a clot iysis system consisting of purified glu-plasminogen, tissue-type plasminogen activator, plasminogen activator inhibitor (released from endothelial cells or blood platelets), fibrinogen, 125T-fibrinogen and thrombin. All proteins were of human origin.In this system APC could increase fibrinolysis in a dose dependent way, without affecting fibrin formation or fibrin crosslinking. However, this profibrinolytic effect of APC could only be observed when plasminogen activator inhibitor (PAI-l) was present. The effect of APC was completely quenched by pretreatment of APC with anti-protein C IgG or di-isopropylfluorophosphate. Addition of the cofactors of APC:protein S, Ca2+-ions and phospholipid-alone or in combination did not enhance the profibrinolytic effect of APC. These observations indicate that human APC can accelerate in vitro clot lysis by the inactivation of PAI-1 activity. However, the neutralization of PAI-1 by APC is independent of the presence or absence of protein S, phospholipid and Ca2+-ions.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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