PubMed Health⌕ Search

Biomedical subjects

Michael S Brown

Publications and source records attributed to Michael S Brown.

At least 19 recordsLinked to original sources
PubMed2006

Allosteric and orthosteric binding modes of two nonpeptide human gonadotropin-releasing hormone receptor antagonists.

Nonpeptide antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) have been the subject of considerable interest because of their potential as a new class of oral therapeutics for the treatment of sex hormone-dependent diseases and infertility. While many classes of competitive GnRH-R antagonists have been described, we present here the first characterization of an allosteric nonpeptide GnRH-R antagonist. Previously, 5-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ylmethyl)furan-2-carboxylic acid (2,4,6-trimethoxyphenyl)amide (here called Furan-1) had been demonstrated to be a potent GnRH-R antagonist both in vitro and in vivo. Using mutagenesis, the binding sites for Furan-1 and another potent nonpeptide antagonist (NBI-42902) have been mapped and are shown to be adjacent but nonoverlapping. Furan-1 is shown to affect the binding kinetics of radiolabeled peptide agonists as well as radiolabeled NBI-42902, and the kinetic data fit the allosteric ternary complex model. Furan-1 is considerably negatively cooperative with the nonpeptide antagonist and extremely negatively cooperative with the peptide agonist [125I-His5,d-Tyr6]GnRH so that it is nearly indistinguishable from an orthosteric competitive compound. Taken together, these data were used to develop a model of the nonpeptides bound to the GnRH-R binding site consistent with the current data.

Allosteric Regulation↗
PubMed2006

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis.

Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells.

Amino Acid Sequence↗
PubMed2006

Protein sensors for membrane sterols.

Cholesterol is an essential component of animal cell membranes, and its concentration is tightly controlled by a feedback system that operates at transcriptional and posttranscriptional levels. Here, we discuss recent advances that explain how cells employ an ensemble of membrane-embedded proteins to monitor sterol concentrations and adjust sterol synthesis and uptake.

Animals↗
PubMed2006

Sterol-regulated ubiquitination and degradation of Insig-1 creates a convergent mechanism for feedback control of cholesterol synthesis and uptake.

This paper describes a convergent mechanism for the feedback control of cholesterol synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for cholesterol synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.

Animals↗
PubMed2006

Geometric and shading correction for images of printed materials using boundary.

A novel technique that uses boundary interpolation to correct geometric distortion and shading artifacts present in images of printed materials is presented. Unlike existing techniques, our algorithm can simultaneously correct a variety of geometric distortions, including skew, fold distortion, binder curl, and combinations of these. In addition, the same interpolation framework can be used to estimate the intrinsic illumination component of the distorted image to correct shading artifacts. We detail our algorithm for geometric and shading correction and demonstrate its usefulness on real-world and synthetic data.

Algorithms↗
PubMed2006

Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin.

Insig-1 and Insig-2 are regulatory proteins that restrict the cholesterol biosynthetic pathway by preventing proteolytic activation of SREBPs and by enhancing degradation of HMG-CoA reductase. Here, we created Insig-double-knockout (Insig-DKO) mice that are homozygous for null mutations in Insig-1 and Insig-2. After 18.5 days of development, 96% of Insig-DKO embryos had defects in midline facial development, ranging from cleft palate (52%) to complete cleft face (44%). Middle and inner ear structures were abnormal, but teeth and skeletons were normal. The animals were lethargic and runted; they died within 1 day of birth. The livers and heads of Insig-DKO embryos overproduced sterols, causing a marked buildup of sterol intermediates. Treatment of pregnant mice with the HMG-CoA reductase inhibitor lovastatin reduced sterol synthesis in Insig-DKO embryos and reduced the pre-cholesterol intermediates. This treatment ameliorated the clefting syndrome so that 54% of Insig-DKO mice had normal faces, and only 7% had cleft faces. We conclude that buildup of pre-cholesterol sterol intermediates interferes with midline fusion of facial structures in mice. These findings have implications for the pathogenesis of the cleft palate component of Smith-Lemli-Opitz syndrome and other human malformation syndromes in which mutations in enzymes catalyzing steps in cholesterol biosynthesis produce a buildup of sterol intermediates.

Animals↗
PubMed2006

Water-vapor detection using asynchronous THz sampling.

The use of a fiber-coupled terahertz (THz) transmitter/receiver pair for spectroscopic detection of water vapor is investigated. Transmission signals of an alumina cylinder demonstrate that the measurement approach can be applied in a windowless ceramic combustor. First, a conventional commercial transmitter/receiver pair is used to make measurements for frequencies to 1.25 THz. Water-vapor absorption is clearly evident within the alumina transparency window and is readily modeled using existing databases. A variety of data-acquisition schemes is possible using THz instrumentation. To assess signal-collection techniques, a prototype THz transmitter/receiver pair is then used with the asynchronous optical-sampling (ASOPS) technique to obtain asynchronous THz-sampling signals to 1 THz without the need for an optomechanical delay line. Two mode-locked Ti:sapphire lasers operating at slightly different repetition rates are used for pumping the transmitter and receiver independently to permit a complete time-domain THz signal to be recorded. The resulting repetitive phase walkout is demonstrated by collecting power spectra of room air that exhibit water-vapor absorption.

Absorption↗
PubMed2006

Effect of dietary lipoic acid on metabolic hormones and acute-phase proteins during challenge with infectious bovine rhinotracheitis virus in cattle.

OBJECTIVE: To determine the effect of dietary supplemental lipoic acid (LA) on serum concentrations of metabolic hormones and acute-phase proteins of steers challenged with infectious bovine rhinotracheitis virus (IBRV). ANIMALS: 32 steers. PROCEDURES: Steers were randomly assigned to 4 treatments: negative control (NC; no LA, no IBRV challenge), control (CON; no LA, IBRV challenge), 16 mg of LA/kg of body weight (BW)/d plus IBRV challenge (LA16), and 32 mg of LA/kg of BW/d plus IBRV challenge (LA32). Following a 21-day adaptation period, CON, LA16, and LA32 steers received IBRV (2 mL/nostril [day 0]); NC steers received saline (0.9% NaCl) solution. Blood samples, nasal swab specimens, BW, and rectal temperatures were obtained 0, 1, 3, 5, 7, 14, and 21 days after challenge. Serum was analyzed for concentrations of haptoglobin, amyloid-A, leptin, and anti-IBRV antibodies. RESULTS: Steers fed LA32 began gaining BW by day 7, whereas BW of CON and LA16 steers declined. Serum haptoglobin concentration of LA32 steers was lower than that of CON and LA16 steers on day 7. Serum neutralization titers for 30 of 32 steers were negative for anti-IBRV antibodies before challenge; however, all steers (including NCs) had antibodies on day 21. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that LA supplementation augmented certain aspects of the immune response; LA32 steers had a more rapid recovery from IBRV viral challenge than did others.

Acute-Phase Proteins↗
PubMed2005

Schoenheimer effect explained--feedback regulation of cholesterol synthesis in mice mediated by Insig proteins.

End-product feedback inhibition of cholesterol synthesis was first demonstrated in living animals by Schoenheimer 72 years ago. Current studies define Insig proteins as essential elements of this feedback system in mouse liver. In cultured cells, Insig proteins are required for sterol-mediated inhibition of the processing of sterol regulatory element-binding proteins (SREBPs) to their nuclear forms. We produced mice with germline disruption of the Insig2 gene and Cre-mediated disruption of the Insig1 gene in liver. On a chow diet, these double-knockout mice overaccumulated cholesterol and triglycerides in liver. Despite this accumulation, levels of nuclear SREBPs and mRNAs for SREBP target genes in lipogenic pathways were not reduced. Whereas cholesterol feeding reduced nuclear SREBPs and lipogenic mRNAs in wild-type mice, this feedback response was severely blunted in the double-knockout mice, and synthesis of cholesterol and fatty acids was not repressed. The amount of HMG-CoA reductase protein was elevated out of proportion to the mRNA in the double-knockout mice, apparently owing to the failure of cholesterol to accelerate degradation of the enzyme. These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in liver as they do in cultured cells.

Alleles↗
PubMed2005

Insig required for sterol-mediated inhibition of Scap/SREBP binding to COPII proteins in vitro.

When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles.

Amino Acid Sequence↗
PubMed2005

Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication.

We recently reported that Hepatitis C virus (HCV) RNA replication requires one or more geranylgeranylated host proteins. Using a combination of [(3)H]mevalonate labeling, coimmunoprecipitation, and bioinformatic search, we identified a geranylgeranylated host protein required for HCV RNA replication. This protein, FBL2, contains an F box domain and a CAAX motif (CVIL). It forms a stable immunoprecipitable complex with the HCV nonstructural protein 5A (NS5A). The association of FBL2 with NS5A requires the CAAX motif of FBL2, but not the F box. Deletion of the F box created a dominant-negative protein that inhibited replication of HCV RNA when overexpressed in Huh7-K2040 cells; this inhibition was overcome by coexpression of NS5A. siRNA-mediated knockdown of FBL2 mRNA by 70% in Huh7-HP cells reduced HCV RNA by 65%; this reduction was overcome by expression of a cDNA encoding a wobble mutant of FBL2. The current data indicate that geranylgeranylated FBL2 binds to NS5A in a reaction crucial for HCV RNA replication.

Cell Cycle Proteins↗
PubMed2005

Combustion exhaust measurements of nitric oxide with an ultraviolet diode-laser-based absorption sensor.

A diode-laser-based sensor has been developed for ultraviolet absorption measurements of the nitric oxide (NO) molecule. The sensor is based on the sum-frequency mixing (SFM) of the output of a tunable, 395-nm external-cavity diode laser and a 532-nm diode-pumped, frequency-doubled Nd:YAG laser in a beta-barium borate crystal. The SFM process generates 325 +/- 75 nW of ultraviolet radiation at 226.8 nm, corresponding to the (v' = 0, v" = 0) band of the A2Sigma+-chi2II electronic transition of NO. Results from initial laboratory experiments in a gas cell are briefly discussed, followed by results from field demonstrations of the sensor for measurements in the exhaust streams of a gas turbine engine and a well-stirred reactor. It is demonstrated that the sensor is capable of fully resolving the absorption spectrum and accurately measuring the NO concentration in actual combustion environments. Absorption is clearly visible in the gas turbine exhaust even for the lowest concentrations of 9 parts per million (ppm) for idle conditions and for a path length of 0.51 m. The sensitivity of the current system is estimated at 0.23%, which corresponds to a detection limit of 0.8 ppm in 1 m for 1000 K gas. The estimated uncertainty in the absolute concentrations that we obtained using the sensor is 10%.

Journal Article↗
PubMed2005

Intramembrane aspartic acid in SCAP protein governs cholesterol-induced conformational change.

The polytopic membrane protein SCAP transports sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi, thereby activating cholesterol synthesis. Cholesterol accumulation in the ER membranes changes SCAP to an alternate conformation in which it binds ER retention proteins called Insigs, thereby terminating cholesterol synthesis. Here, we show that the conserved Asp-428 in the sixth transmembrane helix of SCAP is essential for SCAP's dissociation from Insigs. In transfected hamster cells, mutant SCAP in which Asp-428 is replaced by alanine (D428A) remained in an Insig-binding conformation when cells were depleted of sterols. As a result, mutant SCAP failed to dissociate from Insigs, and it failed to carry SREBPs to the Golgi. These data identify an important functional residue in SCAP, and they provide genetic evidence that the conformation of SCAP dictates the rate of cholesterol synthesis in animal cells.

Amino Acid Sequence↗
PubMed2005

Kinetic behavior of polymer-coated long-period-grating fiber-optic sensors.

A new method of analysis employing the time-dependent response of long-period-grating (LPG) fiber-optic sensors is introduced. The current kinetic approach allows analysis of the time-dependent wavelength shift of the sensor, in contrast to previous studies, in which the LPG sensing element has been operated in an equilibrium mode and modeled with Langmuir adsorption behavior. A detailed kinetic model presented is based on diffusion of the analyte through the outer protective membrane coating into the affinity coating, which is bound to the fiber cladding. A simpler phenomenological approach presented is based on measurement of the slope of the time-dependent response of the LPG sensor. We demonstrate the principles of the kinetic methods by employing a commercial Cu+2 sensor with a carboxymethylcellulose sensing element. The detailed mathematical model fits the time-dependent behavior well and provides a means of calibrating the concentration-dependent time response. In the current approach, copper concentrations below parts per 10(6) are reliably analyzed. The kinetic model allows early-time measurement for low concentrations of the analyte, where equilibration times are long. This kinetic model should be generally applicable to other affinity-coated LPG fiber-optic sensors.

Journal Article↗
PubMed2005

Compensatory increase in fatty acid synthesis in adipose tissue of mice with conditional deficiency of SCAP in liver.

The escort protein SCAP transports SREBPs from ER to Golgi where the active domains are released to activate genes for fatty acid (FA) and cholesterol synthesis. Mice with conditional SCAP deficiency in liver (L-Scap-) manifest marked reductions in hepatic lipid synthesis. Here, we show that the decreased FA synthesis in liver is balanced by an equal increase in nonhepatic tissues, primarily adipose tissue. Extrahepatic synthesis of FAs preserves adipose mass, even when L-Scap- mice consume a fat-free diet. This compensatory response disappears upon fasting, implicating a role for insulin, the major hormonal activator of FA synthesis. This response is mediated by an insulin-dependent increase in adipocyte SREBP-1c and its target mRNAs. In epididymal fat of L-Scap- mice, phosphorylated Akt, Glut-4 mRNA, and glucose uptake are also increased, indicating insulin hypersensitivity. Plasma VLDL triglycerides are dramatically reduced in L-Scap- mice, underscoring the benefits of synthesizing FAs in fat rather than liver.

Adipose Tissue↗
PubMed2004

Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different mechanisms, both involving SCAP and Insigs.

The current paper demonstrates that cholesterol and its hydroxylated derivative, 25-hydroxycholesterol (25-HC), inhibit cholesterol synthesis by two different mechanisms, both involving the proteins that control sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that activate genes encoding enzymes of lipid synthesis. Using methyl-beta-cyclodextrin as a delivery vehicle, we show that cholesterol enters cultured Chinese hamster ovary cells and elicits a conformational change in SREBP cleavage-activating protein (SCAP), as revealed by the appearance of a new fragment in tryptic digests. This change causes SCAP to bind to Insigs, which are endoplasmic reticulum retention proteins that abrogate movement of the SCAP.SREBP complex to the Golgi apparatus where SREBPs are normally processed to their active forms. Direct binding of cholesterol to SCAP in intact cells was demonstrated by showing that a photoactivated derivative of cholesterol cross-links to the membrane domain of SCAP. The inhibitory actions of cholesterol do not require the isooctyl side chain or the Delta5-double bond of cholesterol, but they do require the 3beta-hydroxyl group. 25-HC is more potent than cholesterol in eliciting SCAP binding to Insigs, but 25-HC does not cause a detectable conformational change in SCAP. Moreover, a photoactivated derivative of 25-HC does not cross-link to SCAP. These data imply that cholesterol interacts with SCAP directly by inducing it to bind to Insigs, whereas 25-HC works indirectly through a putative 25-HC sensor protein that elicits SCAP-Insig binding.

Animals↗
PubMed2004

Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain.

Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.

Allosteric Regulation↗