PubMed Health⌕ Search

Biomedical subjects

C Clayton

Publications and source records attributed to C Clayton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structure of a regulatory 3' untranslated region from Trypanosoma brucei.

The African trypanosome Trypanosoma brucei multiplies in mammalian extracellular fluids (bloodstream forms) and in the midgut of Tsetse flies (procyclic forms). The control of gene expression that is necessary to survive in these two environments operates almost exclusively at the posttranscriptional level, and the sequences responsible are located in the 3' untranslated regions of the mRNAs. The major surface proteins of procyclic trypanosomes, EP1, EP2, EP3, and GPEET, are not expressed in bloodstream forms. The 3' untranslated regions of these four mRNAs are not very similar, but all contain a conserved 26mer sequence that is required for developmental regulation. We have analyzed the conformation of the EP1 3' UTR in vitro by enzymatic digestion and lead hydrolysis, and in vivo by modification with DMS and with CMCT (introduced by electroporation). Results indicate that the 3' UTR can be divided into three domains. Domains I and III, at the 5' and 3' ends, form stable structures, but the central domain (domain II), which includes the 26mer, has no stable interactions either within itself, or with other parts of the 3' UTR. Domain I contains three leadzymes that do not conform to the previously reported consensus.

3' Untranslated Regions↗
PubMed1998

Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism.

African trypanosomes compartmentalize glycolysis in a microbody, the glycosome. When growing in the mammalian bloodstream, trypanosomes contain only a rudimentary mitochondrion, and the first seven glycolytic enzymes, including phosphoglycerate kinase, are located in the glycosome. Procyclic trypanosomes, growing in the gut of tsetse flies, possess a fully developed mitochondrion that is active in oxidative phosphorylation. The first six glycolytic enzymes are still glycosomal, but phosphoglycerate kinase is now found in the cytosol. We demonstrate here that bloodstream trypanosomes are killed by expression of cytosolic phosphoglycerate kinase. The toxicity depends on both enzyme activity and cytosolic location. One possible explanation is that cytosolic phosphoglycerate kinase creates an ATP-generating shunt in the cytosol, thus preventing full ATP regeneration in the glycosome and ultimately inhibiting the first, ATP-consuming, steps of glycolysis.

Adenosine Triphosphate↗
PubMed1998

Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.

Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Delta mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles.

Amino Acid Sequence↗
PubMed1998

Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription.

In trypanosomes, most genes are arranged in polycistronic transcription units. Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation. Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting.

Animals↗
PubMed1997

Mechanisms of developmental regulation in Trypanosoma brucei: a polypyrimidine tract in the 3'-untranslated region of a surface protein mRNA affects RNA abundance and translation.

Salivarian trypanosomes are extracellular parasites of mammals that are transmitted by tsetse flies. The procyclic acidic repetitive proteins (PARPs) are the major surface glycoproteins of the form of Trypanosoma brucei that replicates in the fly. The abundance of PARP mRNA and protein is very strongly regulated, mostly at the post-transcriptional level. The 3'-untranslated regions of two PARP genes are of similar lengths, but are dissimilar in sequence apart from a 16mer stem-loop that stimulates translation and a 26mer polypyrimidine tract. Addition of either of these PARP 3'-untranslated regions immediately downstream of a reporter gene resulted in developmental regulation mimicking that of PARP. We show that the PARP 3'-UTR reduces RNA stability and translation in bloodstream forms and that the 26mer polypyrimidine tract is necessary for both effects.

Animals↗
PubMed1997

Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei.

We previously described a system for exogenous control of gene expression in procyclic trypanosomes which depends upon the binding of a tetracycline-inducible repressor to operators situated at the transcriptional start site of the PARP promoter. The recombinant constructs are introduced into non-transcribed spacers of the ribosomal RNA repeat, in an orientation opposite to that of rRNA transcription. Using this system, gene expression could be regulated over four orders of magnitude, but it was not possible to express toxic gene products because selection of recombinant trypanosomes depended on the activity of the inducible promoter. We describe here the characteristics of vectors that include two promoters: a tetracycline-inducible one to drive expression of the toxic products, and a constitutive one to drive transcription of the selectable marker. Relatively high levels of non-induced (non-tetracycline-dependent) expression were seen in some trypanosome clones; this was not usually due to read-through of multiple tandemly-integrated plasmids or tet operator mutations. A variety of constructs differing in resistance marker, 3'-untranslated region (3'-UTR) and the nature of the constitutive promoter was tested. Vectors allowing the successful expression of toxic and other genes in both life cycle stages with regulation factors of up to 700 fold were obtained.

Animals↗
PubMed1997

Reproducibility of Leeds TOR(MAM) mammographic test object plates.

Image quality test objects are essential tools for quality assurance in any branch of radiology. A major problem in test object construction is to make all examples of the test object closely comparable so that results from different examples can be compared. In mammography, the TOR(MAM) test object is considered to be the most sensitive of those generally available in the UK. This paper reports a comparison of 19 examples of this test object design, using five film scorers. Small differences in mean score were found to be statistically significant in about half of the test object plates tested. However, none of the plates differed from the overall mean at the 95% confidence level. Mean differences of score between single plates did not exceed 8%.

Humans↗
PubMed1997

Social drinking and laughter.

Pairs of observers monitored drinking and laughter in groups of social drinkers (N = 56). A significant correlation of .27 was found between units of alcohol consumed and laughter scores.

Adolescent↗
PubMed1997

Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas.

Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion. This organelle is host to a variety of intriguing and unique features, including RNA editing. We examined the characteristics of protein import into mitochondria of Trypanosoma brucei. Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids. The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent. Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes. The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria. Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids). Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms. These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes.

Amino Acid Sequence↗
PubMed1996

The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context.

African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.

Animals↗
PubMed1996

Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.

Aminopterin↗
PubMed1996

Child life programs in the pediatric emergency department.

Child life (CL) programs have long been utilized in the inpatient setting to help in relieving the stress and anxiety associated with hospitalization. The objective of this study was to determine the current availability of such programs in pediatric emergency departments (ED) and what interventions were provided in one ED with a CL program. We conducted a telephone survey of EDs in 44 large children's hospitals in the United States as to their use of child life specialists (CLS). Also, a retrospective review was performed of the CL intervention in one large pediatric ED over a two-year period. Only 6/44 EDs (14%) had at least one full-time CLS, with three of those having two full-time workers. In these EDs the CLS prepared children for painful or anxiety-provoking procedures and provided developmentally appropriate activities in the ED. Another 6/44 (14%) had CL services available by consult. There were no CLS available in 32/44 (72%) EDs, where nursing staff prepared children for painful procedures. EDs with a higher census were more likely to have a full-time CLS (P = 0.02). Hospitals with certified pediatric trauma centers were less likely to have any CL services available in the ED (P = 0.04). A review of the records of the ED at Arkansas Children's Hospital revealed that 32% of patient contacts by the CLS were for invasive procedure teaching and support. Another 22% of interventions were for developmental stimulation. Family support (21.5%) and therapeutic intervention (17.5%) were the next most common contacts. Admission teaching accounted for 4%, while preoperative teaching and support accounted for only 3% of the total. There were more patient contacts made by the worker on evening shift. CL programs, while common in the inpatient setting, are currently underutilized in most pediatric EDs. CLS can provide a variety of services in the pediatric ED.

Adaptation, Psychological↗