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Biomedical subjects

B Middleton

Publications and source records attributed to B Middleton.

At least 55 records · Page 3Linked to original sources
PubMed1994

Modulating the potency of an activator in a yeast in vitro transcription system.

The intrinsic stimulatory potential or potency of a eukaryotic gene activator is controlled by the interaction between the activation domain and the transcriptional machinery. To further understand this interaction, we undertook a biochemical study to identify parameters that could be used to modulate activator potency. We considered how varying the number of activation domains, their flexibility, and the number of promoter sites affects potency in a yeast nuclear extract. The effects of GAL4 derivatives bearing either one, two, or four herpes simplex virus VP16 activation domains (amino acids 413 to 454) were measured on DNA templates containing one or two GAL4 sites in a Saccharomyces cerevisiae nuclear extract. We found that multimerized VP16 activation domains acted synergistically to increase the potency of the activators. The spacing between the activation domains was critical, such that the increased flexibility imparted by a protein linker contributed to increased activator potency. With highly potent activators, the levels of transcription stimulated on a single site were saturating, whereas the stimulatory effect of weaker activators increased with the number of sites. We discuss how these biochemical studies relate to the mechanism of gene activation and synergy in a yeast in vitro system.

Cell Nucleus↗
PubMed1993

The inhibition of cholesterol esterification by cyclandelate in transformed mouse macrophages.

Cyclandelate (trimethylcyclohexanyl mandelate) inhibited cholesterol esterification in a transformed mouse macrophage cell line (J774) with a concentration of approximately 20 microM being required for half-maximal inhibition. The intact drug was required for its inhibitory action since neither of its hydrolysis products, trimethylcyclohexanol and mandelic acid, caused any inhibition even at high concentrations. The drug entered the cells very rapidly with inhibition being apparent within the shortest time possible to measure esterification (15 min after drug addition). The rate of cholesterol esterification returned to control values when drug-inhibited cells were incubated in drug-free medium indicating a rapid loss of drug from the cells. Loading of cells with cholesterol had no effect on the inhibitory action of cyclandelate, and the inhibition of esterification of cholesterol appeared to be specific, since the syntheses of phospholipid and triacylglycerol (which also involve the action of acyltransferases) were not affected by the drug. Similar inhibitions of cholesterol esterification were seen in four other cell lines, a human osteosarcoma, Chinese hamster ovary cells, a human transformed macrophage cell line (U937) and human umbilical cord vein endothelial cells, as well as in slices of pig aorta, indicating a general action in extra-hepatic tissues where the drug is not hydrolysed.

Animals↗
PubMed1993

Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency--diagnosis, plasma carnitine fractions and management in a further patient.

Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD), the third enzyme of the mitochondrial beta-oxidation pathway, carries out the dehydrogenation of 3-hydroxyacyl-CoA compounds of 12-18 carbon length. To date only nine cases of LCHAD deficiency have been documented. We report a further patient who as a neonate developed non-specific gastrointestinal symptoms and at 5 months of age cardiomyopathy, recurrent hypoketotic hypoglycaemia and gross alterations of plasma carnitine fractions. Dietary management with medium chain triglycerides led rapidly to clinical improvement. There was a close correlation between the clinical condition, plasma carnitine fractions and cardiac function. At 2 years of age she is developing normally.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1993

Antihypertensive medication-taking. Investigation of a simple regimen.

In search of strategies to improve compliance, we assessed medication-taking behavior among 19 ambulatory hypertensives, using both pill counts and electronic monitoring. The regimen consisted of one pill each day for < or = 63 weeks with return visits at 1 to 4 week intervals. The study population was 79% male, 68% white, and 16% black with mean (+/- SEM) age 58.4 +/- 2.5 years. Only 51% of the intervisit intervals displayed > or = 80% of vial openings within the desirable range (24 +/- 6 h). Pill counts detected only 2% of suboptimal interdosing intervals identified by electronic monitoring. Early changes in compliance correlated well with later changes (r = 0.83, P = .002). A few of the subjects exhibited a large deviation from the prescription, uncorrectable with drugs having a long duration of action. We conclude that (a) pill counts tend to overestimate patients' compliance rates; (b) changes in medication-taking behavior early in therapy may predict subsequent compliance rates; and (c) prolonging drug action may compensate for some imperfect medication-taking behavior.

Adult↗
PubMed1993

Evaluating consensus among physicians in medical knowledge base construction.

This study evaluates inter-author variability in knowledge base construction. Seven board-certified internists independently profiled "acute perinephric abscess", using as reference material a set of 109 peer-reviewed articles. Each participant created a list of findings associated with the disease, estimated the predictive value and sensitivity of each finding, and assessed the pertinence of each article for making each judgment. Agreement in finding selection was significantly different from chance: seven, six, and five participants selected the same finding 78.6, 9.8, and 1.6 times more often than predicted by chance. Findings with the highest sensitivity were most likely to be included by all participants. The selection of supporting evidence from the medical literature was significantly related to each physician's agreement with the majority. The study shows that, with appropriate guidance, physicians can reproducibly extract information from the medical literature, and thus established a foundation for multi-author knowledge base construction.

Abscess↗
PubMed1992

Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1992

Cyclic AMP stimulates the synthesis and function of the low-density lipoprotein receptor in human vascular smooth-muscle cells and fibroblasts.

1. Cyclic AMP-elevating agents stimulate low-density lipoprotein (LDL) receptor activity in human vascular smooth-muscle cells by increasing the rate of receptor protein synthesis. The stimulation is not secondary to the decrease in the regulatory pool of free cholesterol, since it is unaffected, or even enhanced, by inhibition of cholesterol synthesis and esterification, or inhibition of the conversion of cholesterol into its repressor metabolites. The cyclic AMP-mediated up-regulation of the receptor is maintained at low concentrations of inhibitory sterols, but is eventually over-ridden at high concentrations of these sterols. 2. Cyclic AMP-elevating agents also stimulate the hydrolysis of lysosomal cholesterol esters, thus increasing the cellular cholesterol pool and repressing the expression of the LDL receptor. This cholesterol-mediated repressive effect of cyclic AMP can be prevented by chloroquine, which inhibits lysosomal actions, or by ketoconazole, which inhibits conversion of free cholesterol into its repressor metabolite. Thus the cyclic AMP stimulation of the LDL receptor can be masked by the rapid mobilization of free cholesterol from existing cholesterol esters within cultured cells. 3. We have observed that elevated cyclic AMP concentrations will up-regulate the LDL receptor in cholesterol-depleted human vascular smooth-muscle cells, skin fibroblasts and foetal-lung fibroblasts. We propose that our results are evidence for a cyclic AMP-stimulated, sterol-independent, control of LDL-receptor synthesis which is of widespread occurrence in human cells.

Cells, Cultured↗
PubMed1992

Lysosome inhibitors enhance the ability of cyclic AMP-elevating agents to induce the LDL receptor in human vascular smooth muscle cells.

In human vascular smooth muscle cells cyclic AMP elevation by forskolin increases synthesis of the LDL receptor by a mechanism which appears independent of sterol control. This increased receptor synthesis is further enhanced by chloroquine. Both forskolin and prostaglandin E1 increase the number of cell surface LDL receptors indicating that prostaglandins could exert physiological control over LDL metabolism. This effect is enhanced synergistically by chloroquine. The stimulation by forskolin of LDL receptor synthesis and expression leads to increased metabolism of apo-B and increased hydrolysis of LDL-borne cholesteryl ester. These effects of cyclic AMP on the activity of the LDL pathway are enhanced more than additively by preincubation with the reversible lysosomal inhibitor NH4Cl. Thus cyclic AMP causes up-regulation of the LDL receptor pathway resulting in increased rates of LDL metabolism but this effect can be damped or masked in cell culture by a cyclic AMP-sensitive lysosomal event, probably the acute stimulation of lysosomal cholesterol ester hydrolase.

Alprostadil↗
PubMed1992

Differential diagnosis of hydroxydicarboxylic aciduria based on release of 3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts.

Intact cultured fibroblasts from patients with deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase release 3H2O from [9,10-3H]myristic acid and [9,10-3H]palmitic acid more slowly than normal. The ratio of activity (palmitate/myristate) is also low and the expression (rate with palmitate2/(rate with myristate) gives good differentiation between affected and unaffected cells. In some patients who have shown hydroxydicarboxylic aciduria when unwell there is reduced 3H2O production from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts but normal 3-hydroxyacyl-CoA dehydrogenase activities in disrupted cells. The palmitate/myristate ratio is higher than in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The basic defect in these patients is still unknown but it is suggested that caution be used over the administration of medium-chain triglyceride.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Reuse of knowledge represented in the Arden syntax.

Knowledge Data Systems is building a medical expert system for monitoring clinical events. This system uses the Arden syntax as a knowledge representation. Having encoded may different types of rules in the Arden syntax, we have noticed a number of shortcomings of the syntax. Many of these shortcomings originate from Arden's procedural orientation, from its failure to separate factual medical knowledge from knowledge of how the medical facts should be applied to a particular clinical situation. The absence of this separation leads to redundancy of knowledge and to difficulties in knowledge reuse. We suggest that standards for representing medical logic preserve this separation to engender knowledge reuse. We propose a general framework for representing medical logic which supports both knowledge sharing and reuse.

Diagnosis, Computer-Assisted↗
PubMed1991

Effects of hypothyroidism and high-fat feeding on mRNA concentrations for the low-density-lipoprotein receptor and on acyl-CoA:cholesterol acyltransferase activities in rat liver.

1. Induction of hypothyroidism in rats by feeding propylthiouracil (PTU) significantly increased serum cholesterol concentrations, and the effect was more pronounced for cholesterol in low-density lipoproteins (LDL) rather than high-density lipoproteins (HDL). The concentrations of serum triacylglycerol were decreased in hypothyroidism. These effects on serum lipids were also seen when the normal rats were pair-fed with the PTU-treated group. 2. Feeding a diet rich in saturated fat and cholesterol further increased cholesterol concentrations in LDL and also elevated that in very-low-density lipoprotein (VLDL) of hypothyroid rats. In euthyroid rats such a diet resulted in a relatively small increase in VLDL cholesterol, whereas LDL cholesterol was decreased. 3. Steady-state concentrations of mRNA for the hepatic LDL receptor were significantly decreased in the livers of hypothyroid rats, but were not significantly changed by high-fat feeding in euthyroid or hypothyroid rats. 4. The expression of the LDL receptor in hepatocytes cultured from hypothyroid rats was decreased relative to the euthyroid controls. 5. Whereas the esterification of cholesterol with oleate in hepatocytes cultured from hypothyroid rats was decreased, the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the livers of these animals was not changed. 6. High-fat feeding increased the hepatic ACAT activity in normal and hypothyroid rats. 7. Incubation of rat hepatocytes with 10 nM-tri-iodothyronine for 4 h increased the relative concentration of the mRNA for the LDL receptor by 25%. 8. It is therefore concluded that thyroid hormones stimulate the synthesis and expression of the hepatic LDL receptor. Elevated cholesterol concentrations in LDL in hypothyroidism probably result from a primary defect in the expression of the hepatic receptor, rather than indirectly via changes in ACAT activity.

Animals↗
PubMed1991

Characterization of the LDL receptor in rat promegakaryoblasts in culture.

Rat promegakaryoblasts (RPM, a precursor platelet cell line) in culture exhibited a capacity to bind, take up and degrade 125I-LDL. The low density lipoprotein (LDL) binding showed the following characteristics: (a) high affinity, (b) saturability, (c) specificity, (d) down-regulation, after exposure to 25 hydroxycholesterol. Furthermore the proteolytic degradation of 125I-LDL by RPMs was inhibited by chloroquine which interferes with the lysosomal degradation processes. These findings show LDL receptor cell biology of RPM to be of the classical type and to differ from that of platelets.

Animals↗
PubMed1991

Urinary excretion of 2-methylacetoacetate, 2-methyl-3-hydroxybutyrate and tiglylglycine after isoleucine loading in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency.

The concentrations of 2-methylacetoacetate, 2-methyl-3-hydroxybutyrate and tiglylglycine were determined by gas chromatography-mass spectrometry in urine collected before and for 8 h after loading with 100 mg of isoleucine per kg of body weight. The sum of 2-methylacetoacetate and 2-butanone, a decarboxylation product, was determined as the 2-butanone dinitrophenylhydrazone derivative. Substantial increases in each compound were encountered in a patient with a documented defect of 2-methylacetoacetyl-CoA thiolase. Increased quantities of 2-methyl-3-hydroxybutyrate and tiglylglycine were also found in four children with clinical symptoms similar to those associated with 2-methylacetoacetyl-CoA thiolase deficiency but in whom the activity of the enzyme was found to be normal. The concentration of 2-methylacetoacetate plus 2-butanone in the urine increased after an isoleucine load only in the patient with 2-methylacetoacetyl-CoA thiolase deficiency.

Acetoacetates↗