Search PubMed⌕ Search

Biomedical subjects

S Eaton

Publications and source records attributed to S Eaton.

At least 91 records · Page 5Linked to original sources
PubMed1995

An HPLC assay for sn-1,2-diacylglycerol.

A method for the analysis of 1,2-diacylglycerols in biological samples is presented. After tissue extraction and derivatisation with 3,5-dinitrobenzoyl chloride, samples are analysed by normal phase HPLC, using a 3.9 x 300 mm microPorasil column, and ultraviolet detection at 254 nm. The method gives quantitative recovery of 1,2-diacylglycerol, and is of sufficient sensitivity to allow quantitation of 1,2-diacylglycerol in human muscle needle biopsy specimens, from as little as 10 mg muscle. Human skeletal muscle from fasted control subjects was found to have a 1,2-diacylglycerol content of 455 +/- 78 nmol/g wet weight. The method is robust, giving intra- and inter-assay coefficients of variation of 2.9% and 5.9%, respectively, and should prove useful for the analysis of 1,2-diacylglycerol levels in human disease states, such as diabetes, in which no measurements of 1,2-diacylglycerol have yet been undertaken.

Animals↗
PubMed1995

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium.

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.

Actins↗
PubMed1994

Redox control of beta-oxidation in rat liver mitochondria.

Coupled rat liver mitochondria were incubated with [U-14C]hexadecanoate and carnitine which resulted in the formation of acyl-, 2-enoyl- and 3-hydroxyacyl-CoA and carnitine esters. The production of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters was associated with a significant lowering of the NAD+/NADH ratio, in contrast to rat muscle mitochondria [Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M. & Bartlett, K. (1993) Biochem. J. 289, 161-172], suggesting that control by the respiratory chain is important under normal conditions. When NAD+/NADH ratios were held low by succinate-induced reverse electron flow, 3-enoyl-CoA esters were also detected, probably formed by the action of 3,2-enoyl-CoA isomerase. Measurement of the flux of beta-oxidation at different osmolalities showed that flux was strongly dependent on osmolality changes in the physiological range. Measurement of the CoA and carnitine esters resulting from incubations made at different osmolalities showed that there was an increase in the amounts of the saturated acyl-CoA esters with respect to 2-enoyl-CoA and 3-hydroxyacyl-CoA esters, consistent with control by the electron-transfer flavoprotein-ubiquinone segment [Halestrap, A. P. & Dunlop, J. L. (1986) Biochem. J. 239, 559-565]. This however could not be the only factor operating as indicated by the continued presence of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters at high osmolalities.

Acyl Coenzyme A↗
PubMed1994

Blistered: a gene required for vein/intervein formation in wings of Drosophila.

We have characterized the blistered (bs) locus phenotypically, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. Mosaic analyses show that severe bs alleles behave largely autonomously; homozygous patches having vein-like properties. Developmental analyses were undertaken using light and electron microscopy of wild-type and bs wings as well as confocal microscopy of phalloidin- and laminin-stained preparations. bs defects were first seen early in the prepupal period with the failure of apposition of dorsal and ventral wing epithelia. Correspondingly, during definitive vein/intervein differentiation in the pupal period (18-36 hours after puparium formation), the extent of dorsal/ventral reapposition is reduced in bs wings. Regions of the wing that fail to become apposed differentiate properties of vein cells; i.e. become constricted apically and acquire a laminin-containing matrix basally. To further understand bs function, we examined genetic interactions between various bs alleles and mutants of two genes whose products have known functions in wing development. (i) rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for specification of vein cell fate. rhove mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation. (ii) The integrin genes, inflated and myospheroid, are expressed in intervein cells and are required for adhesion between the dorsal and ventral wing surfaces. Loss of integrin function results in intervein blisters. Integrin mutants interact with bs mutants to increase the frequency of intervein blisters but do not typically enhance vein defects. Both developmental and genetic analyses suggest that the bs product is required during metamorphosis for the initiation of intervein development and the concomitant inhibition of vein development.

Animals↗
PubMed1993

The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

5-Hydroxytryptophan↗
PubMed1993

5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor.

Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes. However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods. As shown recently by Ross et al. (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor. Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA. In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA. Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer. The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA. Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers. The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.

5-Hydroxytryptophan↗
PubMed1993

Intramitochondrial control of the oxidation of hexadecanoate in skeletal muscle. A study of the acyl-CoA esters which accumulate during rat skeletal-muscle mitochondrial beta-oxidation of [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine.

1. We describe the acyl-CoA and acyl-carnitine esters which arise from the incubation of well-coupled State 3 rat skeletal-muscle mitochondrial fractions with [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine. 2. Acyl-CoA ester intermediates of chain length 16, 14, 12, 10 and 8 carbons were detected. 3. Although incubations were in steady state in respect of oxygen consumption, 14CO2 production and generation of acid-soluble radioactivity, quantitative analysis of acyl-CoA esters showed that steady state was not achieved in respect of all intermediates. 4. 3-Hydroxyacyl- and 2-enoyl-CoA and -carnitine esters were found under normoxic conditions. 5. Direct measurement of NAD+ and NADH shows that under identical incubation conditions our observations cannot be explained by gross perturbation of the [NAD+]/[NADH] ratio. 6. We hypothesize that there is a small pool of rapidly recycling NAD+ channelled between complex I of the respiratory chain and the newly described mitochondrial-inner-membrane-associated beta-oxidation trifunctional enzyme [Uchida, Izai, Orii and Hashimoto (1992) J. Biol. Chem. 267, 1034-1041].

Acyl Coenzyme A↗
PubMed1993

External quality assessment in water microbiology: statistical analysis of performance.

A UK-based scheme of water microbiology assessment requires participants to record counts of relevant organisms. Not every sample will contain the target number of organisms because of natural variation and therefore a range of results is acceptable. Results which are tail-end (i.e. at the extreme low or high end of this range) could occasionally be reported by any individual laboratory by chance. Several tail-end results might imply a laboratory problem. Statistical assessment is done in two stages. A non-parametric test of the distribution of tail-end counts amongst laboratories is performed (Cochran's Q) and, if they are not random, then observed and expected frequencies of tail-end counts are compared to identify participants who may have reported excessive numbers of low or high results. Analyses so far have shown that laboratories find high counts no more frequently than would be expected by chance, but that significant clusters of low counts can be detected among participants. These findings have been observed both in short-term and in long-term assessments, thus allowing detection of new episodes of poor performance and intermittent problems. The analysis relies on an objective definition of tail-end results. Working definitions are presented which should identify poor performance in terms of microbiological significance, and which allow fair comparison between membrane-filtration and multiple-tube techniques. Smaller differences between laboratories, which may be statistically significant, will not be detected. Different definitions of poor performance could be incorporated into future assessments.

Colony Count, Microbial↗
PubMed1993

The relationship between the St. Thomas and Oswestry disability scores and the severity of low back pain.

OBJECTIVE: To investigate the relationship between the two disability questionnaires and low back pain severity using the visual analog scale (VAS). DESIGN: One hundred sixty-two chiropractic patients with low back pain were interviewed using an interactive microcomputer system. The sources of data were the St. Thomas and Oswestry disability questionnaires for disability and a VAS for pain severity. All were inherent in the computer interview system. SETTING: The computer review system was set up in the Anglo-European College of Chiropractic outpatient clinic. PATIENTS, PARTICIPANTS: One hundred sixty-two consenting new patients from the general community suffering from low back pain who attended the Anglo-European College of Chiropractic outpatient clinic were asked to participate. MAIN OUTCOME MEASURES: Main outcome measures were the St. Thomas and Oswestry disability questionnaires for disability and the VAS for low back pain severity. RESULTS: There was a moderate correlation between the scores from the St. Thomas and Oswestry disability questionnaires (r = .77, p < .0001). A low but significant correlation score was found when comparing the St. Thomas and Oswestry disability score with pain severity (0.38, p < .0001, and 0.47, p < .0001, respectively). CONCLUSION: The consequence of pain (disability) appears to have a weak relationship to pain severity. Despite the moderate correlation between the two disability questionnaires, it is suggested that they are not interchangeable.

Disability Evaluation↗
PubMed1992

The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation.

cDNAs were isolated that represent transcripts of the Drosophila segment polarity gene, hedgehog (hh). Sequence analysis reveals a motif characteristic of a transmembrane domain, suggesting that the hh protein is membrane-associated. hh expression in epidermal cells is confined to the posterior compartments and coincides precisely with that of engrailed (en). Despite the similar patterns of expression in the cellular blastoderm, hh expression is independent of en, but hh expression becomes sensitive to and dependent on en during the extended germ band stage. The ectopic expression of hh that is normally induced in patched (ptc) mutant embryos does not appear in ptc en double mutants. We discuss these findings in terms of the relationship between en and hh, and the role of the hh function.

Amino Acid Sequence↗
PubMed1992

Pharmacokinetic behavior of gadoteridol injection.

RATIONALE AND OBJECTIVES: To assess the safety and pharmacokinetics of gadoteridol injection (0.5 M) in 18 healthy male volunteers in a phase I clinical trial. METHODS: Volunteers were assigned to one of six dosing groups: 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 mmol/kg gadoteridol (0.5 M), in an ascending dose study. Physical examination, vital signs, electrocardiogram, clinical laboratory tests, and serum and urine samples were obtained at selected time points before and after administration of gadoteridol. RESULTS AND CONCLUSIONS: No significant changes in vital signs, physical examination, clinical laboratory values, or electrocardiogram, that were believed by the principal investigator to be related to the administration of the contrast agent, were observed. A single adverse event (transient hive) believed to be related to contrast agent administration was observed in one volunteer. Pharmacokinetic data show that the elimination half-life and the distribution half-life were independent of the dose used. The mean distribution half-life was 0.20 +/- 0.04 hours, the mean elimination half-life was 1.57 +/- 0.08 hours, and greater than 94% of the drug was excreted in the urine in 24 hours.

Adolescent↗
PubMed1991

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Novel protein-DNA interactions associated with increased immunoglobulin transcription in response to antigen plus interleukin-5.

Although much has been learned about basal levels of immunoglobulin (Ig) transcription, the regulatory effects of cytokines and antigen (Ag) upon Ig expression in lymphocytes have not been fully characterized. We previously reported that Ag plus interleukin-5 (IL-5) caused increased steady-state Ig mRNA levels in Ag-specific cell lines. In this study, we have identified a region between -250 and -125 bp 5' of the Ig transcription start site that is necessary for the induction of increased mu mRNA levels by Ag plus IL-5. Mobility shift and UV cross-linking studies indicated that IL-5 plus Ag induced increased protein binding to this region. Furthermore, this sequence was found to be closely related to another A + T-rich sequence at -525 bp 5' of the transcription start site. Both sequences exhibited similar B-cell-specific and inducible protein binding. Our data suggest that treatment with IL-5 plus Ag induces several DNA-binding proteins, some of which may participate in increasing Ig transcription above basal levels by binding to sequences 5' of the octamer motif.

Animals↗