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Biomedical subjects

M S Brown

Publications and source records attributed to M S Brown.

At least 19 recordsLinked to original sources
PubMed2001

Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene.

Site-1 protease (S1P) cleaves membrane-bound sterol regulatory element-binding proteins (SREBPs), allowing their transcription-stimulating domains to translocate to the nucleus where they activate genes governing lipid synthesis. S1P is a potential target for lipid-lowering drugs, but the effect of S1P blockade in animals is unknown. Here, we disrupt the S1P gene in mice. Homozygous germ-line disruptions of S1P were embryonically lethal. To disrupt the gene inducibly in liver, we generated mice homozygous for a floxed S1P allele and heterozygous for a transgene encoding Cre recombinase under control of the IFN-inducible MX1 promoter. When IFN was produced, 70-90% of S1P alleles in liver were inactivated, and S1P mRNA and protein were reduced. Nuclear SREBPs declined, as did mRNAs for SREBP target genes. Cholesterol and fatty acid biosynthesis in hepatocytes declined by 75%. Low density lipoprotein (LDL) receptor mRNA declined by 50%, as did the clearance of (125)I-labeled LDL from plasma, but plasma cholesterol fell, suggesting that LDL production was reduced. These data raise the possibility that S1P inhibitors may be effective lipid-lowering agents, but they suggest that nearly complete inhibition will be required.

Animals↗
PubMed2001

Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR.

Sterol regulatory element-binding protein-1c (SREBP-1c) enhances transcription of genes encoding enzymes of unsaturated fatty acid biosynthesis in liver. SREBP-1c mRNA is known to increase when cells are treated with agonists of liver X receptor (LXR), a nuclear hormone receptor, and to decrease when cells are treated with unsaturated fatty acids, the end products of SREBP-1c action. Here we show that unsaturated fatty acids lower SREBP-1c mRNA levels in part by antagonizing the actions of LXR. In cultured rat hepatoma cells, arachidonic acid and other fatty acids competitively inhibited activation of the endogenous SREBP-1c gene by an LXR ligand. Arachidonate also blocked the activation of a synthetic LXR-dependent promoter in transfected human embryonic kidney-293 cells. In vitro, arachidonate and other unsaturated fatty acids competitively blocked activation of LXR, as reflected by a fluorescence polarization assay that measures ligand-dependent binding of LXR to a peptide derived from a coactivator. These data offer a potential mechanism that partially explains the long-known ability of dietary unsaturated fatty acids to decrease the synthesis and secretion of fatty acids and triglycerides in livers of humans and other animals.

Animals↗
PubMed2001

SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation.

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting-refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions.

Animals↗
PubMed2001

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes.

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Animals↗
PubMed2001

Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands.

The current paper describes a line of cultured rat hepatoma cells (McA-RH7777 cells) that mimics the behavior of rat liver by producing an excess of mRNA for sterol regulatory element-binding protein 1c (SREBP-1c) as opposed to SREBP-1a. These two transcripts are derived from a single gene by use of alternative promoters that are separated by many kilobases in the genome. The high level of SREBP-1c mRNA is abolished when cholesterol synthesis is blocked by compactin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase that inhibits cholesterol synthesis. Levels of SREBP-1c mRNA are restored by mevalonate, the product of the HMG CoA reductase reaction, and by ligands for the nuclear hormone receptor LXR, including 22(R)-hydroxycholesterol and T0901317. These data suggest that transcription of the SREBP-1c gene in hepatocytes requires tonic activation of LXR by an oxysterol intermediate in the cholesterol biosynthetic pathway. Reduction of this intermediate lowers SREBP-1c levels, and this in turn is predicted to lower the rates of fatty acid biosynthesis in liver.

Animals↗
PubMed2001

Supernumerary orbital muscle in congenital eyelid retraction.

PURPOSE: Although several reports of supernumerary orbital muscles related to the levator palpebrae superioris have been published, no case has been associated with congenital eyelid retraction. This report describes an apparent causal relationship between an accessory levator muscle slip and congenital eyelid retraction. METHODS: Case report and literature review. RESULTS: Release of the anomalous muscle's attachment from the superior tarsal border alone resulted in resolution of the eyelid retraction. CONCLUSIONS: Eyelid muscle anomalies may be a cause of congenital eyelid retraction. Ophthalmologists who treat eyelid disorders should be aware of this possibility when evaluating and operating on patients with congenital eyelid retraction.

Child, Preschool↗
PubMed2001

Patient-specific models for lung nodule detection and surveillance in CT images.

The purpose of this work is to develop patient-specific models for automatically detecting lung nodules in computed tomography (CT) images. It is motivated by significant developments in CT scanner technology and the burden that lung cancer screening and surveillance imposes on radiologists. We propose a new method that uses a patient's baseline image data to assist in the segmentation of subsequent images so that changes in size and/or shape of nodules can be measured automatically. The system uses a generic, a priori model to detect candidate nodules on the baseline scan of a previously unseen patient. A user then confirms or rejects nodule candidates to establish baseline results. For analysis of follow-up scans of that particular patient, a patient-specific model is derived from these baseline results. This model describes expected features (location, volume and shape) of previously segmented nodules so that the system can relocalize them automatically on follow-up. On the baseline scans of 17 subjects, a radiologist identified a total of 36 nodules, of which 31 (86%) were detected automatically by the system with an average of 11 false positives (FPs) per case. In follow-up scans 27 of the 31 nodules were still present and, using patient-specific models, 22 (81%) were correctly relocalized by the system. The system automatically detected 16 out of a possible 20 (80%) of new nodules on follow-up scans with ten FPs per case.

Automation↗
PubMed2001

Spiral versus electron-beam CT for coronary artery calcium scoring.

PURPOSE: To determine differences in coronary artery calcium detection, quantification, and reproducibility, as measured at electron-beam computed tomography (CT) and subsecond spiral CT with retrospective electrocardiogram gating in an asymptomatic adult population. MATERIALS AND METHODS: Seventy subjects asymptomatic for coronary heart disease underwent both electron-beam CT and subsecond spiral CT. In all subjects, two images each were obtained with both scanners. Two experienced readers using three different algorithms scored each of the four scans: one score for the electron-beam CT images and two scores for the spiral CT images. RESULTS: With a 130-HU threshold for the quantification of calcium, there were no significant differences in interscan and interobserver variation in calcium scores between the electron-beam CT and spiral CT images. There was greater interobserver (P <.001) and interscan (P <.03) variation in scores when a 90-HU threshold was used for spiral CT images. With a 130-HU threshold, when calcium scores were used for clinical risk stratification, there was a significant difference between the results obtained with electron-beam CT and those obtained with spiral CT (P <.05). CONCLUSION: Spiral CT has not yet proved to be a feasible alternative to electron-beam CT for coronary artery calcium quantification. There are systematic differences between calcium scores obtained with single-detector array subsecond spiral CT and those obtained with electron-beam CT.

Adult↗
PubMed2001

Semantic processing in visual word recognition: activation blocking and domain specificity.

Lexical decision to a target is typically facilitated when a related prime is simply read, but is typically eliminated if subjects carry out a letter search on the prime when probe and prime appear simultaneously. Three experiments involving the addition of a task-irrelevant word to this prime search paradigm address (1) Stolz and Besner's (1996, 1998) account in which semantic priming is eliminated because letter search on the prime instantiates an activation block between lexical and semantic levels of representation, (2) Chiappe, Smith, and Besner's (1996) account that activation blocking is domain specific, and (3) Neely and Kahan's (2001) claim that semantic activation is context independent and capacity free. The results are (1) consistent with the account that activation blocking is general to a level, rather than item specific, (2) consistent with the account that activation blocking is domain specific, and (3) inconsistent with the claim that semantic activation is context independent and capacity free.

Association↗
PubMed2001

Strabismus due to flap tear of a rectus muscle.

PURPOSE: To present a previously unreported avulsion-type injury of the rectus muscle, usually the inferior rectus, and detail its diagnosis and operative repair. METHODS: Thirty-five patients underwent repair of flap tears of 42 rectus muscles. The muscle abnormality was often subtle, with narrowing or thinning of the remaining attached global layer of muscle. The detached flap of external (orbital) muscle was found embedded in surrounding orbital fat and connective tissue. Retrieval and repair were performed in each case. RESULTS: Fourteen patients had orbital fractures, 7 had blunt trauma with no fracture, and 9 had suspected trauma but did not undergo computed tomographic scan. Five patients experienced this phenomenon following retinal detachment repair. Diagnostically, the predominant motility defect in 25 muscles was limitation toward the field of action of the muscle, presumably as a result of a tether created by the torn flap. These tethers simulated muscle palsy. Seventeen muscles were restricted away from their field of action, simulating entrapment. The direction taken by the flap during healing determined the resultant strabismus pattern. All patients presenting with gaze limitation toward an orbital fracture had flap tears. The worst results following flap tear repair were seen in patients who had undergone orbital fracture repair before presentation, patients who had undergone previous attempts at strabismus repair, and patients who experienced the longest intervals between the precipitating event and the repair. The best results were obtained in patients who underwent simultaneous fracture and strabismus repair or early strabismus repair alone. CONCLUSIONS: Avulsion-type flap tears of the extraocular muscles are a common cause of strabismus after trauma, and after repair for retinal detachment. Early repair produces the best results, but improvement is possible despite long delay.

Adolescent↗
PubMed2000

Unsaturated fatty acids down-regulate srebp isoforms 1a and 1c by two mechanisms in HEK-293 cells.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that increase the synthesis of fatty acids as well as cholesterol in animal cells. All three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from membranes. Previous data indicate that the SREBPs are also negatively regulated by unsaturated fatty acids, but the mechanism is uncertain. In the current experiments, unsaturated fatty acids decreased the nuclear content of SREBP-1, but not SREBP-2, in cultured human embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty acids increased with increasing chain length and degree of unsaturation. Oleate, linoleate, and arachidonate were all effective, but the saturated fatty acids palmitate and stearate were not effective. Down-regulation occurred at two levels. The mRNAs encoding SREBP-1a and SREBP-1c were markedly reduced, and the proteolytic processing of these SREBPs was inhibited. When SREBP-1a was produced by a cDNA expressed from an independent promoter, unsaturated fatty acids reduced nuclear SREBP-1a without affecting the mRNA level. There was no effect when the cDNA encoded a truncated version that was not membrane-bound. When administered together, sterols and unsaturated fatty acids potentiated each other in reducing nuclear SREBP-1. In the absence of fatty acids, sterols did not cause a sustained reduction of nuclear SREBP-1, but they did reduce nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as sterols, can down-regulate nuclear SREBPs and that unsaturated fatty acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c, whereas sterols have their greatest inhibitory effects on SREBP-2.

Base Sequence↗
PubMed2000

Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

Animals↗
PubMed2000

Overexpression of membrane domain of SCAP prevents sterols from inhibiting SCAP.SREBP exit from endoplasmic reticulum.

SCAP (SREBP cleavage-activating protein) forms a complex with sterol regulatory element-binding proteins (SREBPs) and escorts them from the endoplasmic reticulum (ER) to the Golgi complex where proteases release transcriptionally active segments of SREBPs, which enter the nucleus to activate lipid synthesis. The NH(2)-terminal segment of SCAP contains eight transmembrane helices, five of which (TM2-6) comprise the sterol-sensing domain. This domain responds to sterols by causing the SCAP.SREBP complex to be retained in the ER, preventing proteolytic release and reducing transcription of lipogenic genes. Here, we use transfection techniques to overexpress a segment of SCAP containing transmembrane helices 1-6 in hamster and human cells. This segment does not interfere with SCAP.SREBP movement to the Golgi in the absence of sterols, but it prevents sterols from suppressing this movement. This block is abolished when SCAP(TM1-6) contains a point mutation (Y298C) that is known to abolish the activity of the sterol-sensing domain. We interpret these findings to indicate that sterols cause the SCAP.SREBP complex to bind to an ER retention protein through an interaction that involves the sterol-sensing domain. The SCAP(TM1-6) segment competes with the SCAP.SREBP complex for binding to this putative retention protein, thereby liberating the SCAP.SREBP complex so that it can move to the Golgi despite the presence of sterols. These studies provide a potential mechanistic explanation for the ability of sterols to block SCAP.SREBP movement from the ER and thereby to control lipid synthesis in animal cells.

Animals↗
PubMed2000

Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulatory element-binding proteins.

Through suppressive subtractive hybridization, we identified a new gene whose transcription is induced by sterol regulatory element-binding proteins (SREBPs). The gene encodes acetyl-CoA synthetase (ACS), the cytosolic enzyme that activates acetate so that it can be used for lipid synthesis or for energy generation. ACS genes were isolated previously from yeast, but not from animal cells. Recombinant human ACS was produced by expressing the cloned cDNA transiently in human cells. After purification by nickel chromatography, the 701-amino acid cytosolic enzyme was shown to function as a monomer. The recombinant enzyme produced acetyl-CoA from acetate in a reaction that required ATP. As expected for a gene controlled by SREBPs, ACS mRNA was induced when cultured cells were deprived of sterols and repressed by sterol addition. The pattern of regulation resembled the regulation of enzymes of fatty acid synthesis. ACS mRNA was also elevated in livers of transgenic mice that express dominant-positive versions of all three isoforms of SREBP. We conclude that ACS mRNA, and hence the ability of cells to activate acetate, is regulated by SREBPs in parallel with fatty acid synthesis in animal cells.

Acetate-CoA Ligase↗
PubMed2000

Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes.

SREBPs exit the ER in a complex with SCAP. Together, they move to the Golgi where SREBP is cleaved, releasing a fragment that activates genes encoding lipid biosynthetic enzymes. Sterols block ER exit, preventing cleavage, decreasing transcription, and achieving feedback control of lipid synthesis. Here, we report an in vitro system to measure incorporation of SCAP into ER vesicles. When membranes were isolated from sterol-depleted cells, SCAP entered vesicles in a reaction requiring nucleoside triphosphates and cytosol. SCAP budding was diminished in membranes from sterol-treated cells. Kinetics of induction of budding in vitro matched kinetics of ER exit in living cells expressing GFP-SCAP. These data localize the sterol-regulated step to budding of SCAP from ER and provide a system for biochemical dissection.

Animals↗