Kooperativer Bibliotheksverbund

Language: English

In: Molecular and biochemical parasitology, 2011, Vol.175(2), pp.205-208

Description: Very little is known about cell cycle-dependent regulation of mRNA in Trypanosoma brucei, the causative agent of African sleeping sickness. Methods to synchronize cell cycle progression are inefficient or subject the parasites to non-physiological conditions and stress. We developed a fluorescence-activated cell sorting-based method to analyze steady-state mRNA levels in individual cell cycle phases. Normalization of the data was the most challenging problem because internal standards for cell cycle-regulated genes are not available for trypanosomes. Hence, we introduced an external standard (so-called “spike”) to compensate for technically derived variations in processing cells and RNA samples. Validation of this method with a limited number of genes unraveled a transient up-regulation during S and G2/M phases for all mRNAs analyzed. ; Includes references ; p. 205-208.

Keywords: Cell Cycle ; Trypanosoma Brucei ; Bsf ; Mrna Expression ; Pcf ; Quantitative Real Time Pcr ; Fluorescence-Activated Cell Sorting ; Qpcr ; Bloodstream Form ; Procyclic Form ; Facs

ISSN: 0166-6851

URL: View full text (authentication may be required)

Language: English

In: Molecular & Biochemical Parasitology, February 2011, Vol.175(2), pp.205-208

Description: ▶ A reliable protocol to quantify cell-cycle dependent mRNA levels. ▶ Development and validation of an external standard for normalization. ▶ Transient up-regulation of a set of mRNAs during S and G2/M phase. Very little is known about cell cycle-dependent regulation of mRNA in , the causative agent of African sleeping sickness. Methods to synchronize cell cycle progression are inefficient or subject the parasites to non-physiological conditions and stress. We developed a fluorescence-activated cell sorting-based method to analyze steady-state mRNA levels in individual cell cycle phases. Normalization of the data was the most challenging problem because internal standards for cell cycle-regulated genes are not available for trypanosomes. Hence, we introduced an external standard (so-called “spike”) to compensate for technically derived variations in processing cells and RNA samples. Validation of this method with a limited number of genes unraveled a transient up-regulation during S and G2/M phases for all mRNAs analyzed.

Keywords: Qpcr ; Trypanosoma Brucei ; Cell Cycle ; Mrna Expression ; FACS ; Biology ; Chemistry ; Zoology

ISSN: 0166-6851

E-ISSN: 1872-9428

DOI: 10.1016/j.molbiopara.2010.10.001

URL: View record in ScienceDirect (Access to full text may be restricted)

In: PLoS Pathogens, 2016, Vol.12(2)

Description: Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes. Author Summary Trypanosoma brucei is a member of a large group of flagellated protozoan parasites that threatens the lives and husbandry of millions of people worldwide. This group includes parasites that cause devastating diseases such as leishmaniasis (caused by different species of Leishmania ) and Chagas disease in South America (caused by Trypanosoma cruzi ). One common feature of these parasites is a sophisticated life cycle, which requires transmission between a mammalian host and an insect vector. Over the course of this life cycle, the parasites follow a sequence of distinct developmental forms, which are perfectly adapted to the different host environments. We employed quantitative mass spectrometry techniques to unravel the molecular mechanisms that drive developmental differentiation of Trypanosoma brucei , the causative agent of African sleeping sickness. We followed the changes of expression of 4270 proteins during the development of the human-infective to the insect form of trypanosomes. The insights gained should help us to better understand not only how these dangerous parasites are able to survive in different environments, but also how they evade the defense mechanisms of their hosts. Unraveling the requirements needed for adaptation to human hosts might also be useful for targeted drug development to fight the devastating neglected tropical diseases caused by these parasites.

Keywords: Research Article ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Medicine And Health Sciences ; Biology And Life Sciences ; Biology And Life Sciences ; Biology And Life Sciences

ISSN: 1553-7366

E-ISSN: 1553-7374

DOI: 10.1371/journal.ppat.1005439

URL: View source record

Language: English

In: Molecular & cellular proteomics : MCP, January 2013, Vol.12(1), pp.172-9

Description: Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example.

Keywords: Life Cycle Stages ; Dead-Box RNA Helicases -- Metabolism ; Protozoan Proteins -- Metabolism ; Trypanosoma Brucei Brucei -- Growth & Development

ISSN: 15359476

E-ISSN: 1535-9484

DOI: 10.1074/mcp.M112.019224

URL: View this record in MEDLINE/PubMed

Language: English

Description: Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions;however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, post-translational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes.

DOI: https://epub.ub.uni-muenchen.de/38091/1/10.1371_journal.ppat.1005439.pdf

DOI: http://doi.org/10.1371/journal.ppat.1005439

Source: Open Access LMU (Universitätsbibliothek der LMU München)

URL: View full text in LMU