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Biomedical subjects

Y Luo

Publications and source records attributed to Y Luo.

At least 253 records · Page 14Linked to original sources
PubMed1998

Sterol regulatory element binding protein-1 activates the cholesteryl ester transfer protein gene in vivo but is not required for sterol up-regulation of gene expression.

The plasma cholesteryl ester transfer protein (CETP) plays a central role in high density lipoprotein metabolism and reverse cholesterol transport. Plasma CETP levels are increased in response to dietary or endogenous hypercholesterolemia as a result of increased gene transcription in liver and periphery. Deletional analysis in human CETP transgenic mice localized this response to a region of the proximal promoter which contains a tandem repeat of the sterol regulatory element (SRE) of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. The purpose of the present study was to evaluate the role of the SRE-like element in CETP promoter activity. Gel shift assays using CETP promoter fragments containing these elements showed binding of the transcription factors, sterol regulatory element-binding protein-1 (SREBP-1) and Yin Yang-1 (YY-1). Point mutations in the SRE-like element, designated MUT1 and MUT2, resulted in decreased binding of SREBP-1 (MUT1) or SREBP-1 and YY-1 (MUT2). To determine the in vivo significance of this binding activity, CETP transgenic mice were prepared containing these promoter point mutations. MUT1 and MUT2 transgenic mice expressed CETP activity and mass in plasma. In response to high fat, high cholesterol diets, both MUT1-CETP and MUT2-CETP transgenic mice displayed induction of plasma CETP activity similar to that observed in natural flanking region (NFR) CETP transgenic mice. Moreover, in stably transfected adipocyte cell lines, MUT1 and MUT2 CETP promoter-reporter genes showed significant induction of reporter activity in response to sterols. To evaluate transactivation by SREBP-1, NFR- and MUT1-CETP transgenic mice were crossed with SREBP-1 transgenic mice. Induction of the SREBP transgene in the liver with a low carbohydrate diet resulted in a 3-fold increase in plasma CETP activity in NFR-CETP/SREBP transgenic mice, but there was no significant change in activity in MUT1-CETP/SREBP transgenic mice. Thus, SREBP-1 transactivates the NFR-CETP transgene in vivo, as a result of interaction with the CETP promoter SREs. However, this interaction is not required for positive sterol induction of CETP gene transcription. The results suggest independent regulation of the CETP gene by SREBP-1 and a distinct positive sterol response factor.

Animals↗
PubMed1998

Human semaphorin K1 is glycosylphosphatidylinositol-linked and defines a new subfamily of viral-related semaphorins.

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.

Amino Acid Sequence↗
PubMed1998

Intrastriatal injection of dopamine results in DNA damage and apoptosis in rats.

Overflow of the neurotransmitter dopamine (DA) in striatum is implicated in the neurodegenerative processes in ischemia, hypoxia and local exposure to high concentrations of excitatory amino acids. However, how DA causes neurotoxicity is not understood. We report that intrastriatal injection of DA (0.5-1 micromol/microl) in Wistar rats produces a robust increase in apoptotic cell death as determined by both a terminal deoxynucleotidyl transferase catalyzed dUTP-biotin nick labeling (TUNEL) and Klenow polymerase catalyzed [32P]dCTP labeled DNA ladder. Cells in which apoptosis was induced by DA are characterized by condensed chromatin, DNA fragmentation, shrinkage and irregular shapes. The apoptotic cell death induced by DA is not due to the effect of hyperosmolar solution since intrastriatal injection of identical concentrations of NaCl on opposite sides of the same rat brains shows little TUNEL-positive labeling. The number of apoptotic cells is proportional to the amount of DA and length of exposure period. With DA concentrations from 0 to 1 micromol/microl, the maximal toxic effect appears at a concentration of 1 micromol/microl after 24 h exposure. Demonstration of DA-induced apoptosis in vivo may provide a potential molecular mechanism for DA neurotoxicity.

Animals↗
PubMed1998

Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map.

Identification of all human protein-protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein-protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein-protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.

Chromosome Mapping↗
PubMed1998

Type I transforming growth factor beta receptor maps to 9q22 and exhibits a polymorphism and a rare variant within a polyalanine tract.

In a search for mutations of the type I transforming growth factor beta receptor (TbetaR-I), we mapped the gene to 9q22 and found a common polymorphism [TbetaR-I(6A)] and a rare variant [TbetaR-I(10A)] of TbetaR-I, causing an in-frame deletion of three alanines and an in-frame insertion of one alanine, respectively, in the receptor's extracellular domain. The biological relevance of the polymorphism TbetaR-I(6A) was investigated. When TbetaR-I(6A) was transiently transfected into TbetaR-I-deficient cells, the growth-inhibitory effects of transforming growth factor beta were restored. TbetaR-I(6A) and TbetaR-I(10A) frequency were assessed in 108 tumor samples and 80 nontumor samples from patients with a diagnosis of cancer, as well as in 118 normal blood donors of comparable ethnic composition. The frequency of TbetaR-I(6A) heterozygotes was fairly similar in normal blood donors (8%), in nontumor DNA of patients with a diagnosis of cancer (10%), and in tumor samples (14%). However, the frequency of TbetaR-I(6A) homozygotes among nontumor (4%) and tumor (8%) samples obtained from patients with a diagnosis of cancer was higher than that predicted by the Hardy-Weinberg law. The clinical and biological significance of TbetaR-I(6A) homozygosity needs to be further investigated.

Acute Disease↗
PubMed1998

Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells.

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.

Amyloid Precursor Protein Secretases↗
PubMed1998

Two high-resolution crystal structures of the recombinant N-lobe of human transferrin reveal a structural change implicated in iron release.

The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

Animals↗
PubMed1998

Trifluoroethanol stabilizes the pH 4 folding intermediate of sperm whale apomyoglobin.

2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.

Animals↗
PubMed1998

The crystal structure of an intramolecular trans-sialidase with a NeuAc alpha2-->3Gal specificity.

BACKGROUND: Intramolecular trans-sialidase from leech (Macrobdella decora) is a unique enzyme which cleaves the terminal neuraminic acid (NeuAc) residue from sialoglycoconjugates, releasing 2, 7-anhydro-neuraminic acid (2,7-anhydro-NeuAc). It is the first enzyme found to exhibit strictly specific cleavage of NeuAc alpha2-->3Gal linkages in sialoglycoconjugates. The release of 2,7-anhydro-NeuAc instead of NeuAc implies a unique mechanism, in which the sialosyl linkage is transferred within the sialoglycoconjugate rather than hydrolyzed. The aims of the structural study were to gain structural insight into the strict specificity and unique mechanism of this unusual enzyme. RESULTS: . The 2.0 A crystal structure of recombinant leech intramolecular trans-sialidase has been solved by multiple isomorphous replacement. The 1.8 A structure of the enzyme in complex with 2-deoxy-2, 3-didehydro-NeuAc was also solved. The refined model comprising residues 81-769 has a catalytic beta-propeller domain (C), a N-terminal lectin-like domain (II) and an irregular beta-stranded domain (III) inserted into the catalytic domain. The structure reveals several possible carbohydrate-binding features: domain II has a concave face, like that of other sialidases, and there is a suitable surface charge distribution at the domain III-C interface. CONCLUSIONS: Structural comparisons showed closer evolutionary relationships to bacterial sialidases than to viral neuraminidases. Mainchain and sidechain atoms around Thr593 make the glycerol-binding pocket incapable of accommodating an extended equatorial 6-glycerol group, implying that the 6-glycerol group of the reaction intermediate may occupy an axial position, which is also required by the catalytic mechanism. The steric hindrance introduced by the bulky sidechain of Trp734 above the 2-carboxylate group may explain the lack of water involvement in the cleavage reaction and the substrate specificity.

Amino Acid Sequence↗
PubMed1998

Detection of programmed cell death using fluorescence energy transfer.

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.

Amino Acid Sequence↗
PubMed1998

Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway.

Dopamine (DA) is a neurotransmitter, but it also exerts a neurotoxic effect under certain pathological conditions, including age-related neurodegeneration such as Parkinson's disease. By using both the 293 cell line and primary neonatal rat postmitotic striatal neuron cultures, we show here that DA induces apoptosis in a time- and concentration-dependent manner. Concomitant with the apoptosis, DA activates the JNK pathway, including increases in JNK activity, phosphorylation of c-Jun, and subsequent increase in c-Jun protein. This DA-induced JNK activation precedes apoptosis and is persistently sustained during the process of apoptosis. Transient expression of a dominant negative mutant SEK1(Lys --> Arg), an upstream kinase of JNK, prevents both DA-induced JNK activation and apoptosis. A dominant negative c-Jun mutant FLAGDelta169 also reduces DA-induced apoptotic cell death. Anti-oxidants N-acetylcysteine and catalase, which serve as scavengers of reactive oxygen species generated by metabolic DA oxidation, effectively block DA-induced JNK activation and subsequent apoptosis. Thus, our data suggest that DA triggers an apoptotic death program through an oxidative stress-involved JNK activation signaling pathway. Given the fact that the anti-oxidative defense system declines during aging, this molecular event may be implicated in the age-related striatal neuronal cell loss and age-related dopaminergic neurodegenerative disorders, such as Parkinson's and Huntington's diseases.

Animals↗
PubMed1998

Crystallization and preliminary X-ray studies of sialidase L from the leech Macrobdella decora.

Functional monomeric 83 kDa sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from Macrobdella leech, was expressed in Escherichia coli and readily crystallized by a macroseeding technique. The crystal belongs to space group P1 with unit-cell parameters a = 46.4, b = 69.3, c = 72.5 A, alpha = 113.5, beta = 95.4 and gamma = 107.3 degrees. There is one molecule per unit cell, giving a Vm = 2.4 A3 Da-1 and a solvent content of 40%. Native and mercury-derivative data sets were collected to 2.0 A resolution. Threading and molecular-replacement calculations confirmed the existence of a bacterial sialidase-like domain.

Animals↗
PubMed1998

Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.

Adenosine Triphosphatases↗
PubMed1998

Mitochondrial photodamage and PDT-induced apoptosis.

Four photosensitizers with specific targets (mitochondria, lysosomes and plasma membrane) were used to delineate the mechanism of PDT-induced apoptosis in murine leukemia cells. Additional studies were carried out with two sensitizers which caused photodamage to both mitochondria and lysosomes, but varied with regard to membrane photodamage. PDT induced an apoptotic response after mitochondrial photodamage, but not after selective damage to lysosomes or to the cell membrane. Moreover, the latter could delay or inhibit the appearance of apoptosis after mitochondrial photodamage. We had previously reported that exposure of cells to high porphycene concentrations caused an apoptotic response in the dark; this was also associated with mitochondrial damage. These results are consistent with recent proposals that release of mitochondrial components can trigger an apoptotic response. ATP depletion after mitochondrial photodamage does not appear to play a role in initiation of the apoptotic program.

Adenosine Triphosphate↗
PubMed1998

The induction of apoptosis by a positively charged methylene blue derivative.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cell death are to be fully understood. PDT with a methylene blue analog DO15 yielded mitochondrial photodamage whilst membrane and lysosomal integrity were maintained. Apoptosis was detected using the DNA stain HO342, by the appearance of 50 kb fragments and by DNA ladder formation. The release of mono- and oligonucleosomes was further quantified using an ELISA protocol. Large DNA fragments were observed immediately following illumination, and nucleosomes were detected at 1-2 h post-treatment. Increasing the dose 4-fold accelerated the apoptotic response to PDT. This is the first report of a thiazine photosensitiser inducing apoptosis and is consistent with recent proposals suggesting that release of mitochondrial components may play an important role in the mechanism of cell death.

Animals↗
PubMed1998

New methods for three dimensional mapping of brain waves.

The representation of spatiotemporal pattern of brain electrical activity is an important issue in the research of neural assemblies. This paper describes methods of parametric 3D head or brain modeling and its corresponding interpolation for functional imaging based on brain waves. The 3D interpolation method is an extension of cortical imaging technique. It can be used with features of either transformed domains or higher order statistics of brain waves on realistic head or brain models. The simulating result suggests that it is a better method in comparison with the global nearest neighbor technique. With those techniques, an example of brainwave imaging was given using partial coherence of event-related potentials estimated by multivariate autoregressive model.

Brain Mapping↗
PubMed1998

Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment.

Most early-onset familial Alzheimer disease (AD) cases are caused by mutations in the highly related genes presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations produce increases in beta-amyloid (Abeta) formation and apoptosis in many experimental systems. A cDNA (ALG-3) encoding the last 103 amino acids of PS2 has been identified as a potent inhibitor of apoptosis. Using this PS2 domain in the yeast two-hybrid system, we have identified a neuronal protein that binds calcium and presenilin, which we call calsenilin. Calsenilin interacts with both PS1 and PS2 in cultured cells, and can regulate the levels of a proteolytic product of PS2. Thus, calsenilin may mediate the effects of wild-type and mutant presenilins on apoptosis and on Abeta formation. Further characterization of calsenilin may lead to an understanding of the normal role of the presenilins and of the role of the presenilins in Alzheimer disease.

Age of Onset↗