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Biomedical subjects

P Leahy

Publications and source records attributed to P Leahy.

At least 19 recordsLinked to original sources

Extraocular muscle is defined by a fundamentally distinct gene expression profile.

Skeletal muscle fibers are defined by patterned covariation of key traits that determine contractile and metabolic characteristics. Although the functional properties of most skeletal muscles result from their proportional content of a few conserved muscle fiber types, some, typically craniofacial, muscles exhibit fiber types that appear to lie outside the common phenotypic range. We analyzed gene expression profiles of three putative muscle classes, limb, masticatory, and extraocular muscle (EOM), in adult mice by high-density oligonucleotide arrays. Pairwise comparisons using conservative acceptance criteria identified expression differences in 287 genes between EOM and limb and/or masticatory muscles. Use of significance analysis of microarrays methodology identified up to 400 genes as having an EOM-specific expression pattern. Genes differentially expressed in EOM reflect key aspects of muscle biology, including transcriptional regulation, sarcomeric organization, excitation-contraction coupling, intermediary metabolism, and immune response. These patterned differences in gene expression define EOM as a distinct muscle class and may explain the unique response of these muscles in neuromuscular diseases.

Animals↗

Sterol regulatory element-binding protein-1c mimics the negative effect of insulin on phosphoenolpyruvate carboxykinase (GTP) gene transcription.

We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC ) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between -355 and -277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein.

Animals↗

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Adult↗

CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription.

Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.

Binding Sites↗

Molecular and cellular adaptations to carbohydrate and fat intake.

Adaptation to carbohydrate and fat intake involves changes in a number of biochemical parameters at the cellular level. A change in the concentration of fat or carbohydrate in the blood acts directly to influence metabolic pathways by altering the flux of intermediates into cells. This in turn alters the concentration of hormones and other signaling molecules and changes the rate of expression of genes coding for key regulatory proteins or enzymes in metabolic pathways. These effects occur at different rates and in a tissue-specific manner in response to diet. A key metabolic adaptation involves changes in the level of expression of genes coding for proteins of critical importance in energy metabolism; this is largely due to an altered rate of transcription of selected genes under the control of hormones and/or carbohydrate and lipid. The mediators of this effect are transcription factors, that is, nuclear proteins which integrate the effects of hormones and substrates with the transcription process by binding to response elements in the promoters of regulated genes and interacting with the transcription machinery at the TATA box, thereby altering the activity of RNA polymerase II. In this review we will outline the hierarchy of cellular adaptations to diet and will emphasize the latest concepts of gene regulation in response to metabolites and hormones. In particular, we will review the role of the various transcription factors involved in the regulated expression of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.132), a key gluconeogenic enzyme.

Adaptation, Physiological↗

Nuclear factor I regulates expression of the gene for phosphoenolpyruvate carboxykinase (GTP).

Nuclear factor-I (NFI) binds to the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter immediately 5' to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3' of its original location. However if the CRE was moved 5 base pairs 3', placing it out of phase with the other elements in the promoter, or moved 5' to -285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

Animals↗

Reliability of measurements obtained with a modified functional reach test in subjects with spinal cord injury.

BACKGROUND AND PURPOSE: The primary purpose of this study was to determine whether the Functional Reach Test (FRT) could be modified to provide reliable measurements of sitting balance. A secondary purpose was to determine whether the test could be used to measure differences among levels of spinal cord injury. SUBJECTS: Thirty male subjects with spinal cord injuries were divided into into three groups based on injury type. Group 1 consisted of subjects with C5-6 tetraplegia, group 2 consisted of subjects with T1-4 paraplegia, and group 3 consisted of subjects with T10-12 paraplegia. METHODS: Subjects sat on similar mat tables (tables varied based on what was available at a given clinic) against the same backboard, set at 80 degrees. During two sessions, forward reach was measured with a yardstick, with a 10-minute break between sessions. RESULTS: Intraclass correlation coefficients (3,2) were high and varied from .85 to .94. Post hoc testing revealed that differences occurred between groups 1 and 3 and groups 2 and 3, but not between groups 1 and 2. CONCLUSION AND DISCUSSION: Test-retest reliability was high with modification of the FRT with a single rater. The measurements reflected differences among levels of lesion. Further study is needed to determine normal values for all levels of lesion, relationships to functional outcomes, and effects of equipment on sitting balance. The modified FRT appears to provide reliable measurements of sitting balance in nonstanding persons with spinal cord injuries.

Adolescent↗

Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis.

Results of previous studies indicated that treatment of diabetic rats (induced by streptozotocin) with cobalt chloride (CoCl2) resulted in a significant decrement in serum glucose concentration. The present study was designed to determine the potential role of enhanced glucose uptake vs. decreased glucose production in the above response. The rate of systemic appearance of glucose, measured under fasting conditions using [3-3H]glucose tracer, was reduced from 35.5 +/- 2.5 to 17.5 +/- 1.8 micromol . kg-1 . min-1 in diabetic rats treated with 2 mM CoCl2 added to the drinking water for 10-14 days (P < 0.01). Tissue accumulation of intravenously administered 2-deoxy-[14C]glucose was significantly reduced in kidney and eye of diabetic rats treated with CoCl2, whereas the uptake remained unchanged in several other tissues including cerebrum, red and white skeletal muscle, heart, and liver. The relative content of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased 3.1-fold in livers of diabetic compared with normal rats (P < 0.001), and treatment of diabetic rats with CoCl2 decreased hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the liver was decreased by 33% in CoCl2-treated normal rats (P < 0.05). Treatment with CoCl2 resulted in no change in cAMP levels in the livers of either diabetic or normal rats. These results suggest that the glycemia-lowering effect of CoCl2 is mediated by reductions in the rate of systemic appearance of glucose and hepatic gluconeogenesis.

Animals↗

Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers.

A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.

Animals↗

Promoter usage determines tissue specific responsiveness of the rat acetyl-CoA carboxylase gene.

The acetyl-CoA carboxylase gene contains two promoters, PI and PII which generate multiple mRNA forms. We have used the reverse transcription-polymerase chain reaction to investigate tissue specific promoter usage in rats either fed a standard chow diet, starved for 48 h, or starved and then refed a high carbohydrate, low fat diet. Expression of PII-generated mRNAs was seen in all tissues examined and was not dramatically changed by food removal or refeeding. PI-generated mRNAs were expressed at variable levels in a narrower range of tissues and were regulated by these dietary manipulations. Thus only the PI promoter is responsive to diet and the ability of a tissue to use this promoter determines whether it can alter fatty acid synthesis in response to nutritional challenges.

Acetyl-CoA Carboxylase↗

Tissue-specific regulation of lipogenic mRNAs by thyroid hormone.

We have previously shown that triiodothyronine (T3) regulates rat fatty acid synthesis in a tissue specific manner. Here, we determined the effects of thyroid state on mRNAs encoding the lipogenic enzymes, acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS). S14 mRNA, a sequence tightly associated with lipogenesis, was also measured. Levels of the three mRNA were 9-13-fold higher in hyper- than hypothyroid liver. Limited expression in kidney and heart was also increased by thyroid hormone. In brown adipose tissue, highest levels were recorded in hypothyroid animals. Thyroid state did not affect expression in lung and brain. All these changes are consistent with those previously measured in fatty acid synthesis. In white adipose tissue, mRNA expression was increased by hyperthyroidism. This increase may not be reflected in fatty acid synthesis, since we recently showed lipogenesis to be reduced under these circumstances. All three mRNAs responded rapidly to T3 in liver, but more slowly in kidney and fat. Thus, T3 regulates lipogenesis by altering levels of ACC and FAS mRNAs. S14 mRNA changes in parallel.

Acetyl-CoA Carboxylase↗

Regulation of brown adipose tissue lipogenesis by thyroid hormone and the sympathetic nervous system.

Thyroid hormone regulates lipogenesis differently in rat liver and brown adipose tissue (BAT). In the hypothyroid state, lipogenesis is suppressed in liver but enhanced in BAT. Here we investigated the mechanisms underlying increased lipogenesis in hypothyroid BAT. Housing the animals at 28 degrees C decreased lipogenesis in hypothyroid BAT to euthyroid levels. Denervation resulted in a 90% reduction in lipogenesis in hypothyroid BAT such that levels were lower than in euthyroid tissue. Thyroid hormone treatment of hypothyroid rats stimulated fatty acid synthesis in denervated BAT, as in liver, but decreased it in intact BAT. Steady-state levels of mRNA encoding acetyl-CoA carboxylase, fatty-acid synthase, and spor 14 were measured in similar animals by Northern analysis. The expression of these mRNAs mirrored the lipogenic data, showing that both thyroid hormone and the sympathetic nervous system work at a pretranslational level in this tissue. These data suggest that the increased BAT lipogenesis found with hypothyroidism is mediated by the sympathetic nervous system to counter the reduction in metabolic rate in these animals.

Adipose Tissue, Brown↗

Precasting work sheet--an assessment tool. A clinical report.

Physical therapists often use casting as an adjunct to traditional management of patients with brain injury. Casts generally are used on these patients for one of two reasons: 1) to increase range of motion with the use of serial casting or 2) to augment functional treatment with the use of inhibitive casting. This clinical report describes serial and inhibitive casts and introduces a precasting work sheet that promotes the selection of appropriate casting patients and necessary planning. The work sheet guides the therapist through an assessment of patient data relevant to casting, including passive range of motion, postural tone and abnormal reflexes, and voluntary movement. The precasting work sheet helps therapists gain expertise in casting by familiarizing them with the factors to consider before casting, encouraging them to set goals for casting, and helping them to determine whether the goals were met.

Brain Injuries↗

Novel biotinylated plasmid expression vectors retain biological function and can bind streptavidin.

A new method for coupling proteins to plasmid expression vectors is presented. Biotin was covalently attached to a plasmid expression vector containing a chloramphenicol acetyltransferase (CAT) gene. The specific label was one biotin per 100 bp. An electrophoretic mobility shift assay showed that the plasmid was capable of binding multiple streptavidin molecules. When transfected into mouse fibroblasts, the biotinylated plasmid retained 40% of the native plasmid's biological activity, as determined by CAT assay, and was not affected by the binding of streptavidin. The method allows for attachment of any protein to plasmid DNA expression vector while retaining biological function. Hybrid plasmids in which the transcription cassettes were kept free of biotin label were constructed by digesting biotinylated and unbiotinylated plasmids at sites outside the transcription cassette and re-ligating the digestion products. Electron microscopy studies show that the ligation products formed large tangled assemblages of plasmid DNA. When equimolar (with respect to gene number) amounts of these large hybrid biotinylated plasmids were transfected into mouse fibroblasts by means of calcium phosphate precipitation, an increase in CAT expression 25-fold greater than that of original biotinylated plasmid was observed. Slot-blot analysis of total DNA extracted from transfected cells shows that this enhanced activity was not due to increased transfection efficiency. Receptor-mediated delivery could not be shown when a complex comprising biotinylated asialoglycoprotein/streptavidin/biotinylated CAT expression vector was placed in media containing Hep G2 cells.

Animals↗