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C Clayton

Publications and source records attributed to C Clayton.

At least 19 recordsLinked to original sources
PubMed2006

Activation of the caspase 8 pathway mediates seizure-induced cell death in cultured hippocampal neurons.

In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2003

Spinal anaesthesia indirectly depresses cortical activity associated with electrical stimulation of the reticular formation.

BACKGROUND: Neuraxial blockade reduces the requirements for sedation and general anaesthesia. We investigated whether lidocaine spinal anaesthesia affected cortical activity as determined by EEG desynchronization that occurs following electrical stimulation of the midbrain reticular formation (MRF). METHODS: Six goats were anaesthetized with isoflurane, and cervical laminectomy performed to permit spinal application of lidocaine. The EEG was recorded before, during and after focal electrical stimulation (0.1, 0.2, 0.3 and 0.4 mA) in the MRF while keeping the isoflurane concentration constant. RESULTS: During lidocaine spinal anaesthesia, the spectral edge frequency (SEF) after MRF electrical stimulation (13.6 (SD 1.0) Hz, averaged across all stimulus currents) was less than the SEF during control and recovery periods (18.6 (3.6) Hz and 17.2 (2.2) Hz, respectively; P<0.05). Bispectral index values were similarly affected: 69 (10) at control compared with 55 (6) during the spinal block (P<0.05). CONCLUSIONS: These results suggest that lidocaine spinal anaesthesia blocks ascending somatosensory transmission to mildly depress the excitability of reticulo-thalamo-cortical arousal mechanisms.

Anesthesia, Spinal↗
PubMed2002

Composition of the editing complex of Trypanosoma brucei.

The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.

Amino Acid Sequence↗
PubMed2001

Degradation of the unstable EP1 mRNA in Trypanosoma brucei involves initial destruction of the 3'-untranslated region.

Kinetoplastid protozoa regulate their gene expression primarily through control of mRNA degradation and translation. We describe here the degradation of three reporter mRNAs in Trypanosoma brucei. One mRNA had the 3'-untranslated region (3'-UTR) from the developmentally regulated EP1 mRNA, which is abundant in the procyclic (tsetse fly) form of the parasite but is almost undetectable in the bloodstream form. This untranslated region includes a 26 nt U-rich sequence that causes extreme RNA instability in the bloodstream form. The two other RNAs, which are not developmentally regulated, had either the actin 3'-UTR, or a version of the EP1 sequence lacking the 26 nt bloodstream-form instability element. All RNAs had poly(A) tails approximately 200 nt long, in both bloodstream and procyclic forms. Degradation of the two constitutively expressed mRNAs involved deadenylation and degradation by both 5'-->3' and 3'-->5' exonucleases. In contrast, in bloodstream forms, the 3'-end of the RNA bearing the bloodstream-form instability element disappeared very rapidly after transcription inhibition and partially deadenylated intermediates were not seen. The instability element may cause extremely rapid deadenylation, or it may be targeted by an endonuclease.

3' Untranslated Regions↗
PubMed2001

Measurements of radiation near an atomic spectral line from the interaction of a 30 GeV electron beam and a long plasma.

Emissions produced or initiated by a 30-GeV electron beam propagating through a approximately 1-m long heat pipe oven containing neutral and partially ionized vapor have been measured near atomic spectral lines in a beam-plasma wakefield experiment. The Cerenkov spatial profile has been studied as a function of oven temperature and pressure, observation wavelength, and ionizing laser intensity and delay. The Cerenkov peak angle is affected by the creation of plasma, and estimates of neutral and plasma density have been extracted. Increases in visible background radiation, consistent with increased plasma recombination emissions due to dissipation of wakefields, were simultaneously measured.

Journal Article↗
PubMed2001

The exosome of Trypanosoma brucei.

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.

Alleles↗
PubMed2001

Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is metabolized to pyruvate. Aerobic metabolism depends on the activities of glycosomal triosephosphate isomerase and a mitochondrial glycerophosphate oxidase, and on glycerophosphate<-->dihydroxyacetone phosphate exchange across the glycosomal membrane. Using a combination of genetics and computer modelling, we show that triosephosphate isomerase is probably essential for bloodstream trypanosome survival, but not for the insect-dwelling procyclics, which preferentially use amino acids as an energy source. When the enzyme level decreased to about 15% of that of the wild-type, the growth rate was halved. Below this level, a lethal rise in dihydroxyacetone phosphate was predicted. Expression of cytosolic triosephosphate isomerase inhibited cell growth. Attempts to knockout the trypanosome alternative oxidase genes (which are needed for glycerophosphate oxidase activity) were unsuccessful, but when we lowered the level of the corresponding mRNA by expressing a homologous double-stranded RNA, oxygen consumption was reduced fourfold and the rate of trypanosome growth was halved.

Adenosine Triphosphate↗
PubMed2001

The coatomer of Trypanosoma brucei.

Coatomer is a multisubunit complex involved in trafficking of vesicles between the endoplasmatic reticulum and the Golgi apparatus. From sequence homologies, all seven subunits, alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP, are encoded in the genome of Trypanosoma brucei. The complete predicted amino-acid sequences of beta-, beta'-, and zeta-COP show only 20-30% identity with higher eucaryotic homologues. The trypanosome coatomer complex was partially purified using a procedure similar to that used for bovine coatomer.

Amino Acid Sequence↗
PubMed2001

An essential dimeric membrane protein of trypanosome glycosomes.

Kinetoplastid parasites compartmentalize the first seven enzymes of glycolysis in a peroxisome-like microbody, the glycosome. Genes encoding the most abundant protein of the glycosomal membrane, GIM5, have been cloned and the protein characterized. Two genes, GIM5A and GIM5B, encode 26 kDa proteins. Although many microbody membrane proteins are conserved in evolution, the only homologues of GIM5 in the available databases are from the closely related kinetoplastids Trypanosoma cruzi and Leishmania. The N- and C-termini are conserved between the two genes, and between species, and are oriented towards the cytosol. They are separated by a short loop that is located between two transmembrane domains and shows almost no sequence conservation. This suggests that the N- and C-terminal domains are more important for function. GIM5 forms dimers in vivo. Overexpression of GIM5B inhibits growth, whereas depletion of GIM5 to below 10% of wild-type levels is very rapidly lethal. This novel organellar membrane protein is therefore essential for bloodstream trypanosome survival.

Amino Acid Sequence↗
PubMed2000

Binding of coatomer by the PEX11 C-terminus is not required for function.

Microbodies are single membrane-bound organelles found in eukaryotes from trypanosomes to man. Although they have diverse roles in metabolism, the mechanisms and molecules involved in membrane biogenesis and matrix protein import are conserved. Similarly, the basic mechanisms and structures involved in vesicular transport are similar throughout eukaryotic evolution. The PEX11 proteins are required for the division of microbodies in trypanosomes, yeast and mammals, and a role of coatomer in this process has been suggested. We show here that the binding of trypanosome, yeast and bovine coatomers to selected peptides is identical. Coatomer binds to the C-termini of trypanosome PEX11 and rat Pex11alpha, but not yeast Pex11p or human Pex11beta. Mutations of the C-terminus of trypanosome PEX11 that eliminated coatomer binding did not affect function in yeast or trypanosomes. Thus binding of coatomer to the C-terminus of PEX11 is not required for PEX11 function.

Animals↗
PubMed2000

Large-scale analysis of gene expression changes during acute and chronic exposure to [Delta]9-THC in rats.

Large-scale cDNA microarrays were employed to assess transient changes in gene expression levels following acute and chronic exposure to cannabinoids in rats. A total of 24,456 cDNA clones were randomly selected from a rat brain cDNA library, amplified by PCR, and arrayed at high density to investigate differential gene expression profiles following acute (24 h), intermediate (7 days), and chronic (21 days) exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the psychoactive ingredient of marijuana. Hippocampal mRNA probes labeled with (33)P obtained from both vehicle and Delta(9)-THC-treated animals were hybridized with identical cDNA microarrays. Results revealed a total of 49 different genes altered by Delta(9)-THC exposure; of these, 28 were identified, 10 had homologies to expressed sequence tags (ESTs), and 11 had no homology to known sequences in the GenBank database. Chronic or acute cannabinoid receptor activation altered expression of several genes (i.e., prostaglandin D synthase, calmodulin) involved in biochemical cascades of cannabinoid synthesis or cannabinoid effector systems. Other genes [i.e., neural cell adhesion molecule (NCAM), myelin basic protein], whose relation to cannabinoid system function was not immediately obvious, were also significantly altered. Verification of the changes obtained with the large-scale screen was determined by RNA dot blots in different groups of animals treated the same as those in the large-scale screen. Results are discussed in terms of the different types of genes affected at different times during chronic Delta(9)-THC exposure.

Animals↗
PubMed2000

Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress.

In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.

Animals↗
PubMed2000

Screening to the converted: an educational intervention in African American churches.

BACKGROUND: African American women have higher incidences of breast and cervical cancers and African American men present with more advanced stages of colon and prostate cancers than do their non-African American counterparts. Since the church is central to the organization of the African American community, the authors set out to determine whether a church-directed educational project could influence parishioners to obtain cancer screening. METHODS: Three African American churches having memberships of 250, 500, and 1,500, respectively, were selected for their different socioeconomic strata: one congregation was composed mostly of working poor, the second was more affluent, and the third consisted primarily of retirees. During a five-week summer period, appropriate literature, health fairs, testimonials by cancer survivors, and visits by representatives of the medical community were used to increase awareness of cancer screening. Surveys regarding cancer-screening behaviors were distributed at the end of church services. Using the guidelines established by the American Cancer Society, individual recommendations for screening examinations were developed and sent to parishioners based on their survey responses. RESULTS: Of 437 parishioners surveyed (73% female, 27% male), 75% were 40 years old or older. Many reported up-to-date screening for breast (84%), cervical (78%), colon (62%), and prostate (89%) cancers. The results were remarkably similar in all three churches. Telephone follow-up seven months after the survey directed at the 120 parishioners identified as noncompliant for at least one cancer screening revealed that 49% had obtained the appropriate screenings. CONCLUSIONS: These African American churchgoers were well screened compared with estimated national averages, possibly due to previous efforts of the activist ministers in the churches selected. The message for cancer screening is heeded when delivered through the African American church.

Adult↗
PubMed2000

Toxoplasma gondii catalase: are there peroxisomes in toxoplasma?

The intracellular protozoan parasite Toxoplasma gondii, like all members of the phylum Apicomplexa, is known to possess many organelles: in addition to mitochondria and the compartments of the secretory pathway, there is a reduced chloroplast (the apicoplast) and the phylum-specific components of the apical complex: dense granules, micronemes and rhoptries. Conspicuously missing so far are microbodies, organelles that can be found in nearly all eukaryotic organisms. Microbodies show a large variation with regard to their size, number and contents, depending on the organism and cell type. One marker enzyme of this single membrane-bound organelle is catalase, which is responsible for the degradation of hydrogen peroxide to water and oxygen. The EST project in T. gondii revealed the existence of two overlapping clones which showed similarity with catalase, and these were used to clone the corresponding gene. The predicted sequence of T. gondii catalase has -AKM at the C terminus, which falls within the consensus of the PTS1 peroxisomal targeting signal. Southern blot analysis confirmed the presence of a single copy gene. Northern and western blot analyses showed that the catalase gene is transcribed and translated. Immunofluorescence assays using an antibody raised against a catalase peptide identified a distinct structure towards the apical end, but other catalase-specific antibodies failed to confirm this localisation. Cell fractionations indicated that the majority of the enzyme was in the cytosol. The fusion of the C-terminal twelve amino acids, including AKM, or the canonical peroxisomal targeting signal, -SKL, to GFP resulted in predominantly cytosolic localization in T. gondii. There was therefore no evidence for membrane-bound peroxisomes in Toxoplasma.

Amino Acids↗
PubMed1999

Transcription of 'inactive' expression sites in African trypanosomes leads to expression of multiple transferrin receptor RNAs in bloodstream forms.

African trypanosomes express a heterodimeric transferrin receptor that mediates iron uptake from the host bloodstream. The genes encoding the receptor, ESAG6 and ESAG7, are found at the beginning of VSG expression sites: these are telomeric, polycistronic transcription units that each terminate with a gene encoding a trypanosome variant surface glycoprotein, VSG. Approximately 20 of these VSG expression sites are found in the trypanosome genome, but only one VSG is expressed at a time. The conventional view is that one expression site promoter is extremely active whereas the others are either inactive or show very low, poorly processive activity, and that all transferrin receptor molecules are encoded by the active expression site. The 3'-end of the ESAG6 gene is more than 5 kb from the promoter. We show here that 20% of ESAG6 mRNA originates from the 'inactive' expression sites. We suggest that many expression site promoters in trypanosomes show low-level activity throughout the life cycle, and that transcription proceeds for at least 5 kb. This suggests a simplified model of VSG expression site control, whereby the only regulated event is the strong activation of a single expression site promoter in bloodstream forms.

Amino Acid Sequence↗
PubMed1999

The development of within-song type variation in song sparrows.

We investigated the development of within-song type variation in song sparrows, Melospiza melodia, with two experiments designed to determine how exposure to within-type variation influences the song-learning process and whether within-type variation itself is a learned trait. In the first experiment, we compared learning between two groups of males, one group tutored exclusively with song models presented with no variation, and the other group tutored exclusively with song models presented with a range of within-type variation that is normally produced by birds in the field. The two groups in this experiment did not differ significantly in any measure of how well they learned, suggesting that exposure to within-type variation has no measurable influence on the learning process overall. Nor did the groups differ in the expression of within-song type variation in their own adult songs, demonstrating that within-type variation is not a learned feature of song sparrow song. In the second experiment, we tutored a single group of birds with both invariant and variable models, allowing us to ask how within-type variability affects learning preferences. Young birds preferentially copied song type models presented with variation significantly more than invariant models. Taken together, these experiments provide insight into the evolution of within-song type variation in song sparrows, although the functional significance of this level of variation and learning preferences based on variation remain enigmatic. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗