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Biomedical subjects

Min You

Publications and source records attributed to Min You.

At least 19 recordsLinked to original sources
PubMed2006

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain.

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.

Achondroplasia↗
PubMed2006

Transmembrane helix heterodimerization in lipid bilayers: probing the energetics behind autosomal dominant growth disorders.

Here, we show that the energetics of transmembrane helix heterodimer formation can be characterized in liposomes using Förster resonance energy transfer (FRET). We present the theory and the protocol for measuring the free energy of heterodimerization, and the total (hetero and homo-dimeric) dimer fraction. We use the presented methodology to determine the propensity for heterodimer formation between wild-type fibroblast growth factor receptor 3 (FGFR3) transmembrane domain and the Ala391Glu mutant, linked to Crouzon syndrome with acanthosis nigricans.

Acanthosis Nigricans↗
PubMed2005

FGFR3 dimer stabilization due to a single amino acid pathogenic mutation.

Mutations in the transmembrane (TM) domains of receptor tyrosine kinases (RTKs) have been implicated in the induction of pathological phenotypes. These mutations are believed to stabilize the RTK dimers, and thus promote unregulated signaling. However, the energetics behind the pathology induction has not been determined. An example of a TM domain pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer. Here, we determine the free energy of dimerization of wild-type and mutant FGFR3 TM domain in lipid bilayers using Förster resonance energy transfer, and we show that hydrogen bonding between Glu391 and the adjacent helix in the dimer is a feasible mechanism for dimer stabilization. The measured change in the free energy of dimerization due to the Ala391-->Glu pathogenic mutation is -1.3 kcal/mol, consistent with previous reports of hydrogen bond strengths in proteins. This is the first quantitative measurement of mutant RTK stabilization in a membrane environment. We show that this seemingly modest value can lead to a large increase in dimer fraction and thus profoundly affect RTK-mediated signal transduction.

Alanine↗
PubMed2005

Forster resonance energy transfer in liposomes: measurements of transmembrane helix dimerization in the native bilayer environment.

The lipid bilayer vesicle is a model of the cellular membrane. Even in this simple system, however, measuring the thermodynamics of membrane protein association is a challenge. Here we discuss Forster resonance energy transfer (FRET) in liposomes as a method to probe the dimerization of transmembrane helices in a membrane environment. Although the measurements are labor intensive, FRET in liposomes can be measured accurately provided that attention is paid to sample homogeneity and sample equilibration. One must also take into account statistical expectations and the FRET that results from random colocalization of donors and acceptors in the bilayer. Without careful attention to these details, misleading results are easy to obtain in membrane FRET experiments. The results that we obtain in model systems are reproducible and depend solely on the concentration of the protein in the bilayer (i.e., on the protein-to-lipid ratio), thereby yielding thermodynamic parameters that are directly relevant to processes in biological membranes.

Dimerization↗
PubMed2005

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications.

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TMwt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TMKK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TMKK elutes much earlier than TMwt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.

Amino Acid Sequence↗
PubMed2005

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forster resonance energy transfer suggest weak interactions between fibroblast growth factor receptor 3 (FGFR3) transmembrane domains in the absence of extracellular domains and ligands.

Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, fibroblast growth factor receptor 3 (FGFR3), dimerize in lipid vesicles in the absence of the extracellular domains and ligands. We have performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with peptides produced via solid-phase peptide synthesis that correspond to the TM domain of FGFR3. We have carried out Forster resonance energy transfer (FRET) measurements using two donor-acceptor pairs, fluorescein/rhodamine and Cy3/Cy5, as a function of peptide concentration and donor-to-acceptor mole ratios. Our results suggest that FGFR3 TM domains form sequence-specific dimers in lipid bilayers. However, the dimerization propensity of FGFR3 TM domain is much weaker than the dimerization propensity of glycophorin A (GpA), the well-characterized "membrane dimer standard". We discuss our findings in the context of cell signaling across the plasma membrane and diseases or disorders that occur due to single amino acid mutations in the TM domain of FGFR3.

Amino Acid Sequence↗
PubMed2005

Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice.

The protective effect of dietary saturated fatty acids against the development of alcoholic liver disease has long been known, but the underlying mechanism is not completely understood. We examined the involvement of the adipocyte hormone adiponectin. Circulating adiponectin levels were significantly elevated by chronic ethanol administration to mice consuming a diet high in saturated fat. The increase in circulating adiponectin was associated with the activation a set of hepatic signaling pathways mediated through AMP-activated protein kinase, PPAR-alpha, and PPAR-gamma coactivator alpha, which in turn led to markedly increased rates of fatty acid oxidation, prevention of hepatic steatosis, and alleviation of liver enzyme changes. Furthermore, treatment of rat 3T3-L1 adipocytes with saturated fatty acids (palmitic or stearic acids) in the presence of ethanol increased secretion of adiponectin and enhanced activity of a mouse adiponectin promoter. In conclusion, the protective action of saturated fat against the development of alcoholic fatty liver in mice is partially mediated through induction of adiponectin. The present findings suggest a novel paradigm for dietary fatty acids in the pathogenesis of alcoholic liver disease and provide a promising therapeutic strategy-nutritional modulation of adiponectin-in treating human alcoholic fatty liver disease.

3-Hydroxybutyric Acid↗
PubMed2005

Arthrobacter scleromae sp. nov. isolated from human clinical specimens.

A gram-positive, coryneform bacterium was isolated from swollen scleromata of a dermatosis patient. An analysis of its phenotypic, chemotaxonomic, and genotypic characteristics showed that this bacterium is closely associated with Arthrobacter oxydans and Arthrobacter polychromogenes but that it belongs to a distinct species, for which the name Arthrobacter scleromae sp. nov. is proposed.

Adult↗
PubMed2004

Molecular mechanisms of alcoholic fatty liver: role of sterol regulatory element-binding proteins.

Alcoholic fatty liver is the earliest and most common response of the liver to alcohol in heavy alcohol use, and it may be a precursor of more severe forms of liver injury. We and colleagues in our laboratory found that in two rat hepatoma cell lines, H4IIEC3 and McA-RH7777, ethanol markedly induced transcription of a sterol regulatory element-binding protein (SREBP)-regulated promoter through increased levels of mature SREBP-1 protein. Whereas inhibition of ethanol oxidation by 4-methylpyrazole blocked the effect, the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol in the hepatoma cells, supporting the idea that the effect is likely mediated by acetaldehyde. Consistent with these in vitro findings, consumption of a low-fat diet with ethanol by mice for 4 weeks resulted in a significant increase in the abundance of the mature (active) form of hepatic SREBP-1. Activation of SREBP-1 by ethanol feeding was associated with increased expression of lipogenic genes as well as the accumulation of triglyceride in the livers. Taken together, these findings seem to indicate that metabolism of ethanol increased hepatic lipogenesis by activating SREBP-1 and that this effect of ethanol may contribute to the development of alcoholic fatty liver. We and colleagues in our laboratory further studied the mechanisms of ethanol activation of SREBP-1 by identifying a new target of ethanol, adenosine 5'-monophosphate (AMP)-activated protein kinase. Our study results demonstrated that the effect of ethanol on SREBP-regulated promoter activation was mediated, at least in part, through inhibition of AMP-activated protein kinase. Consistent with this hypothesis, chronic ethanol feeding (4 weeks) resulted in a significantly reduced activity and protein level of AMP-activated protein kinase and increased acetyl coenzyme A carboxylase activity in the mouse livers.

Animals↗
PubMed2004

Molecular mechanisms of alcoholic fatty liver: role of peroxisome proliferator-activated receptor alpha.

Normal function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is crucial for the regulation of hepatic fatty acid metabolism. Fatty acids serve as ligands for PPARalpha, and when fatty acid levels increase, activation of PPARalpha induces a battery of fatty acid-metabolizing enzymes to restore fatty acid levels to normal. Hepatic fatty acid levels are increased during ethanol consumption. However, results of in vitro work showed that ethanol metabolism inhibited the ability of PPARalpha to bind DNA and activate reporter genes. This observation has been further studied in mice. Four weeks of ethanol feeding of C57BL/6J mice also impairs fatty acid catabolism in liver by blocking PPARalpha-mediated responses. Ethanol feeding decreased the level of retinoid X receptor alpha (RXRalpha) as well as the ability of PPARalpha/RXR in liver nuclear extracts to bind its consensus sequence, and the levels of mRNAs for several PPARalpha-regulated genes were reduced [long-chain acyl coenzyme A (acyl-CoA) dehydrogenase and medium-chain acyl-CoA dehydrogenase] or failed to be induced (acyl-CoA dehydrogenase, liver carnitine palmitoyl-CoA transferase I, very long-chain acyl-CoA synthetase, very long-chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals. Consistent with this finding, ethanol feeding did not induce the rate of fatty acid beta-oxidation, as assayed in liver homogenates. Inclusion of WY14,643, a PPARalpha agonist, in the diet restored the DNA-binding activity of PPARalpha/RXR, induced mRNA levels of several PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation in liver homogenates, and prevented fatty liver in ethanol-fed animals. Blockade of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by WY14,643.

Animals↗
PubMed2004

The role of AMP-activated protein kinase in the action of ethanol in the liver.

BACKGROUND & AIMS: Our previous work has shown that ethanol induces the fatty acid synthesis pathway by activation of sterol regulatory element-binding protein 1 (SREBP-1). In the present study, we studied the mechanisms of this activation by identifying a new target of ethanol, AMP-activated protein kinase (AMPK). METHODS: The effects of ethanol on AMPK, acetyl-CoA carboxylase (ACC), and SREBP-1 were assessed in rat hepatic cells and in the livers of ethanol-fed mice. RESULTS: In rat hepatoma H4IIEC3 or McA-RH 7777 cell lines, ethanol-induced transcription of an SREBP-regulated promoter was suppressed by the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or metformin, 2 known AMPK activators. Consistent with this, over expression of a constitutively active form of AMPK blocked the effect of ethanol, whereas coexpression of a dominant-negative form of AMPK augmented the effect. Moreover, activation of AMPK by metformin or AICAR largely blocked the ability of ethanol to increase levels of mature SREBP-1 protein. These findings suggest that the effect of ethanol on SREBP-regulated promoter activation was partially mediated through AMPK inhibition. We further demonstrated that AMPK was inhibited by ethanol in hepatic cells. In parallel, ethanol increased the activity of ACC and suppressed the rate of palmitic acid oxidation. Finally, feeding mice a low-fat diet with ethanol resulted in significantly reduced hepatic AMPK activity, increased ACC activity, and enhanced malonyl CoA content. CONCLUSIONS: Taken together, our findings suggest that AMPK may play a key role in regulating the effects of ethanol on SREBP-1 activation, fatty acid metabolism, and development of alcoholic fatty liver.

AMP-Activated Protein Kinases↗
PubMed2004

Overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology.

Alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH2) are responsible for metabolizing the bulk of ethanol consumed as part of the diet and their activities contribute to the rate of ethanol elimination from the blood. They are expressed at highest levels in liver, but at lower levels in many tissues. This pathway probably evolved as a detoxification mechanism for environmental alcohols. However, with the consumption of large amounts of ethanol, the oxidation of ethanol can become a major energy source and, particularly in the liver, interferes with the metabolism of other nutrients. Polymorphic variants of the genes for these enzymes encode enzymes with altered kinetic properties. The pathophysiological effects of these variants may be mediated by accumulation of acetaldehyde; high-activity ADH variants are predicted to increase the rate of acetaldehyde generation, while the low-activity ALDH2 variant is associated with an inability to metabolize this compound. The effects of acetaldehyde may be expressed either in the cells generating it, or by delivery of acetaldehyde to various tissues by the bloodstream or even saliva. Inheritance of the high-activity ADH beta2, encoded by the ADH2*2 gene, and the inactive ALDH2*2 gene product have been conclusively associated with reduced risk of alcoholism. This association is influenced by gene-environment interactions, such as religion and national origin. The variants have also been studied for association with alcoholic liver disease, cancer, fetal alcohol syndrome, CVD, gout, asthma and clearance of xenobiotics. The strongest correlations found to date have been those between the ALDH2*2 allele and cancers of the oro-pharynx and oesophagus. It will be important to replicate other interesting associations between these variants and other cancers and heart disease, and to determine the biochemical mechanisms underlying the associations.

Alcohol Dehydrogenase↗
PubMed2004

Recent advances in alcoholic liver disease II. Minireview: molecular mechanisms of alcoholic fatty liver.

Alcohol has long been thought to cause fatty liver by way of altered NADH/NAD(+) redox potential in the liver, which, in turn, inhibits fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. More recent studies indicate that additional effects of ethanol both impair fat oxidation and stimulate lipogenesis. Ethanol interferes with DNA binding and transcription-activating properties of peroxisome proliferator-activated receptor-alpha (PPARalpha), as demonstrated with cultured cells and in ethanol-fed mice. Treatment of ethanol-fed mice with a PPARalpha agonist can reverse fatty liver even in the face of continued ethanol consumption. Ethanol also activated sterol regulatory element binding protein 1, inducing a battery of lipogenic enzymes. These effects may be due in part to inhibition of AMP-dependent protein kinase, reduction in plasma adiponectin, or increased levels of TNF-alpha in the liver. The understanding of these ethanol effects provides new therapeutic targets to reverse alcoholic fatty liver.

AMP-Activated Protein Kinases↗
PubMed2003

Peroxisome proliferator-activated receptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunction and abnormalities in hepatic lipid metabolism in ethanol-fed mice.

Proper function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is essential for the regulation of hepatic fatty acid metabolism. Fatty acid levels are increased in liver during the metabolism of ethanol and should activate PPARalpha. However, recent in vitro data showed that ethanol metabolism inhibited the function of PPARalpha. We now report that ethanol feeding impairs fatty acid catabolism in the liver in part via blocking PPARalpha-mediated responses in C57BL/6J mice. Ethanol feeding decreased PPARalpha/retinoid X receptor alpha binding in electrophoretic mobility shift assay of liver nuclear extracts. mRNAs for PPAR-regulated genes were reduced (long chain and medium chain acyl-CoA dehydrogenases) or failed to be induced (acyl-CoA oxidase, liver carnitine palmitoyl-CoA transferase, very long chain acyl-CoA synthetase, very long chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals, and ethanol feeding did not increase the rate of fatty acid beta-oxidation. Wy14,643, a PPARalpha agonist, restored the DNA binding activity of PPARalpha/retinoid X receptor alpha, induced mRNA levels of PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation, and prevented fatty liver in ethanol-fed animals. Impairment of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by Wy14,643.

Animals↗
PubMed2003

Identification of a retinoid receptor response element in the human aldehyde dehydrogenase-2 promoter.

BACKGROUND: The human aldehyde dehydrogenase-2 promoter contains sites that bind members of the nuclear receptor family, and one (designated FP330-3') is predicted to bind retinoic acid receptors. METHODS: Binding of retinoid receptors to the FP330-3' oligonucleotide duplex and point mutations thereof was assayed using electrophoretic mobility shift assays. The function of the promoter element was determined in transfection assays. RESULTS: Heterodimers of retinoic acid receptor (RAR)alpha, beta, and gamma with retinoid X receptor (RXR)alpha bound the FP330-3' site. Mutagenesis of the FP330-3' site suggested that either the upstream DR-5 or downstream DR-1 could mediate binding of RAR/RXR. FP330-3' oligonucleotide duplexes were not bound by in vitro translated RXR homodimers but weakly competed with a synthetic DR-1 oligonucleotide duplex for binding by RXR. A reporter construct carrying four copies of the FP330-3' element was induced by cotransfection of rat hepatoma cells with a construct encoding RARalpha, when the RAR-specific ligand AM580 was present. Each of the three RXR isoforms alpha, beta, and gamma stimulated the expression of reporter constructs containing the FP330-3' sites in a 9-cis retinoic acid-dependent fashion in cells in culture. This was confirmed in the case of RXRalpha using the RXR-specific ligand methoprene. CONCLUSION: The human aldehyde dehydrogenase-2 promoter contains a retinoid response element, which may contribute to regulation of the gene.

Aldehyde Dehydrogenase↗
PubMed2002

Reusable platforms for high-throughput on-chip temperature gradient assays.

This paper describes a reusable platform that can apply a linear temperature gradient to a lab-on-a-chip device. When a planar microfluidic device with a series of microchannels is placed on top of the platform with the channels perpendicular to the gradient, each channel is held at a discrete temperature. This allows temperature-dependent data for chemical or biochemical species flowed into the device to be obtained in a concurrent fashion. As a demonstration, a melting curve for dsDNA is performed by collecting all the data simultaneously. The gradient is stable enough to easily distinguish between 30-mers where the complement strand contains a single C-A mismatch or a single T-G mismatch or is a perfect match. On the other hand, a temperature gradient can be formed parallel to the direction of flow of the microchannels. This allows the temperature in each channel to vary continuously as the liquid flows downstream. If each microchannel in the array contains a distinct pH value, ionic strength, species concentration, or chemical composition, then a high-throughput two-variable experiment can be performed. We demonstrate this mode of data collection by measuring the fluorescence yield of fluorescein dye molecules in aqueous solution simultaneously as a function of concentration and temperature.

Journal Article↗
PubMed2002

Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP).

Alcoholic fatty liver is the earliest and most common response of the liver to alcohol and may be a precursor of more severe forms of liver injury. The mechanism by which ethanol causes fatty liver and liver injury is complex. We found that in both rat H4IIEC3 and McA-RH7777 hepatoma cell lines, ethanol induced transcription of a sterol regulatory element-binding protein (SREBP)-regulated promoter via increased levels of mature SREBP-1 protein. This effect of ethanol was blocked by addition of sterols. This effect is likely mediated by acetaldehyde, because the effect was only seen in cell lines expressing alcohol dehydrogenase, and inhibition of ethanol oxidation by 4-methylpyrazole blocked the effect in the hepatoma cells. Furthermore, the aldehyde dehydrogenase inhibitor cyanamide enhanced the effect of ethanol in the hepatoma cells. Consistent with these in vitro findings, feeding mice a low fat diet with ethanol for 4 weeks resulted in a significant increase in steady-state levels of the mature (active) form of SREBP-1. Activation of SREBP-1 by ethanol feeding was associated with increased expression of hepatic lipogenic genes as well as the accumulation of triglyceride in the livers. These finding suggest that metabolism of ethanol increased hepatic lipogenesis by activating SREBP-1 and that this effect of ethanol may contribute to the development of alcoholic fatty liver.

Alcohol Dehydrogenase↗