Search PubMed⌕ Search

Biomedical subjects

S Liang

Publications and source records attributed to S Liang.

At least 37 records · Page 2Linked to original sources
PubMed2001

Proteomic detection of changes in protein synthesis induced by NGX6 transfected in human nasopharyngeal carcinoma cells.

In the previous study, we cloned a new gene, named NGX6, related to nasopharyngeal carcinoma (NPC) at 9p. To study its function in the pathogenesis of NPC, we have investigated changes in protein synthesis between NPC cell line HNE1 and that transfected with the gene. Using high-resolution two-dimensional electrophoresis, we found that 22 protein spots showed variations that were significant and reproducible. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches identified seven proteins that were upregulated and seven proteins that were downregulated. These proteins included Fas, zinc-finger protein (ZNF), RAB, and Ah receptor-interacting protein (AIP). The functional implications of the identified proteins are discussed.

Down-Regulation↗
PubMed2001

Chemical carboxyl-terminal sequence analysis of peptides and proteins using tribenzylsilyl isothiocyanate.

A new derivatization reagent, tribenzylsilyl isothiocyanate (TBS-ITC), is applied to C-terminal peptide and protein sequencing. It has been successfully used to sequence six C-terminal residues of house apomyoglobin and a synthetic peptide at low nanomole levels. The chemistry involves activation with acetic anhydride, derivatization with TBS-ITC, and cleavage of derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. The tribenzylsilyl is a bulky, electric donor group and is a good leaving group. It facilitates the nucleophilic attack of the NCS(-1) in the coupling reaction. The efficiency for C-terminal sequencing by TBS-ITC is about the same as that of acetyl isothiocyanate (AITC), which is a derivatizing reagent for C-terminal sequencing developed by our laboratory. TBS-ITC is much more stable than AITC and trimethylsilyl isothiocyanate (TMS-ITC). TBS-ITC is a solid with relatively long shelf life, whereas AITC and TMS-ITC are liquid and not stable at room temperature.

Amino Acid Sequence↗
PubMed2001

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.

Amino Acid Sequence↗
PubMed2001

Structural analysis of free and enzyme-bound amaranth alpha-amylase inhibitor: classification within the knottin fold superfamily and analysis of its functional flexibility.

The three-dimensional structure of the amaranth alpha-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to alpha-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution.

Algorithms↗
PubMed2001

Analysis of SAW properties of epitaxial ZnO films grown on R-Al2O3 substrates.

ZnO thin films with a high piezoelectric coupling coefficient are widely used for high frequency and low loss surface acoustic wave (SAW) devices when the film is deposited on top of a high acoustic velocity substrate, such as diamond or sapphire. The performance of these devices is critically dependent on the quality of the ZnO films as well as of the interface between ZnO and the substrate. In this paper, we report the studies on piezoelectric properties of epitaxial (1120) ZnO thin films grown on R-plane sapphire substrates using metal organic chemical vapor deposition (MOCVD) technique. The c-axis of the ZnO film is in-plane. The ZnO/R-Al2O3 interface is atomically sharp. SAW delay lines, aligned parallel to the c-axis, were used to characterize the surface wave velocity, coupling coefficient, and temperature coefficient of frequency as functions of film thickness to wavelength ratio (h/lambda). The acoustic wave properties of the material system were calculated using Adler's matrix method, and the devices were simulated using the quasi-static approximation based on Green's function analysis.

Journal Article↗
PubMed2001

Detection of an mRNA polymorphism by differential display.

Differential Display (DD) technology was utilized to compare programs of gene expression in primary cultures of human skin fibroblasts from normal volunteers and patients diagnosed with melancholic depression. Polymorphic transcripts of a single gene differing by one tandem repeat sequence of four nucleotides (TGAT) in the 3' noncoding region were detected.

3' Untranslated Regions↗
PubMed2000

Large scale gene expression analysis of cholesterol-loaded macrophages.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.

Cell Line↗
PubMed2000

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

Alanine Transaminase↗
PubMed2000

The amino-terminal domain of apolipoprotein B does not undergo retrograde translocation from the endoplasmic reticulum to the cytosol. Proteasomal degradation of nascent apolipoprotein B begins at the carboxyl terminus of the protein, while apolipoprotein B is still in its original translocon.

We studied the sequential topology of the NH(2) and COOH termini of apoB during translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 cells, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41% (relative to apoB100) of its amino acid sequence. We conducted similar studies using monoclonal antibodies against the NH(2) and COOH termini of apoB100 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Myc or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucyl-norleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% were isolated by anti-c-Myc antibodies. Proteinase K digestion of total microsomes consistently generated two fragments ( approximately 70 and approximately 120 kDa) of apoB42 containing the NH(2) terminus throughout the chase; no fragments containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling studies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies support a model in which, in the absence of adequate core lipid synthesis, there is partial translocation of apoB leading to cytosolic exposure, ubiquitination, and proteasomal degradation directly from the original translocation channel.

Animals↗
PubMed2000

Construction of protein binding sites in scaffold structures.

We have developed a strategy for grafting a protein-protein interface based on the known crystal structure of a native ligand and receptor proteins in a complex. The key interaction residues at the ligand protein binding interface are transferred onto a scaffold protein so that the mutated scaffold protein will bind the receptor protein in the same manner as the ligand protein. First, our method identifies key residues and atoms in the ligand protein, which strongly interact with the receptor protein. Second, this method searches the scaffold protein for combinations of candidate residues, among which the distance between any two candidate residues is similar to that between relevant key interaction residues in the ligand protein. These candidate residues are mutated to key interaction residues in the ligand protein respectively. The scaffold protein is superposed onto the ligand protein based upon the coordinates of corresponding atoms, which are assumed to strongly interact with the receptor protein. Complementarity between scaffold and receptor proteins is evaluated. Scaffold proteins with a low superposing rms difference and high complementary score are accepted for further analysis. Then, the relative position of the scaffold protein is adjusted so that the interfaces between the scaffold and receptor proteins have a reasonable packing density. Other mutations are also considered to reduce the desolvation energy or bad steric contacts. Finally, the scaffold protein is cominimized with the receptor protein and evaluated. To test the method, the binding interface of barstar, the inhibitor of barnase, was grafted onto small proteins. Four scaffold proteins with high complementary scores are accepted.

Bacillus↗
PubMed2000

Thermal noise reduction of mechanical oscillators by actively controlled external dissipative forces

We show that the thermal fluctuations of very soft mechanical oscillators, such as the cantilever in an atomic force microscope (AFM), can be reduced without changing the stiffness of the spring or having to lower the environment temperature. We derive a theoretical relationship between the thermal fluctuations of an oscillator and an actively controlled external dissipative force. This relationship is verified by experiments with an AFM cantilever where the external active force is coupled through a magnetic field. With simple instrumentation, we have reduced the thermal noise amplitude of the cantilever by a factor of 3.4, achieving an apparent temperature of 25 K with the environment at 295 K. This active noise reduction approach can significantly improve the accuracy of static position or static force measurements in a number of practical applications.

Journal Article↗
PubMed2000

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping.

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3-10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight >10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.

Animals↗
PubMed2000

A quantitative reverse transcription and polymerase chain reaction assay for human IGF-II allows direct comparison of IGF-II mRNA levels in cancerous breast, bladder, and prostate tissues.

Previously, we showed by in situ hybridization that insulin-like growth factor (IGF)-II is upregulated in approximately 50% of prostate, breast, and bladder tumours. In this study, a quantitative competitive reverse transcription and polymerase chain reaction (QC RT-PCR) assay was established and used to quantify human IGF-II mRNA levels in cells and tissues. In this QC RT-PCR assay, a competitor IGF-II RNA, prepared from a newly constructed plasmid encoding the human IGF-II sequence with a 110-bp fragment inserted, was added to RNA samples prior to RT-PCR. The human IGF-II specific QC RT-PCR assay has allowed us to readily compare the levels of IGF-II mRNA in human tissues and cultured cells. Consistent with our previous observations by in situ hybridization, IGF-II mRNA was up-regulated in 50% of cancerous breast tissues examined as compared to the matching benign tissues, and IGF-II mRNA levels were higher in bladder tumours than breast and prostate tumours. In summary, we present here quantitative data confirming that a subclass of breast cancer samples has elevated levels of IGF-II transcripts by the new competitive RT-PCR assay.

Breast Neoplasms↗
PubMed2000

Grafting of protein-protein interaction epitope.

Transferring the biological function of one protein to another is a key issue in understanding the structure and function relationship of proteins. We have developed a strategy for grafting protein-protein interaction epitopes. As a first step, residues at the interface of the ligand protein which strongly interact with the receptor protein were identified. Then protein scaffolds were docked onto receptor protein based on geometric complementarity. Only high docking score matches were saved. For each saved match, the scaffold protein was accepted if it had suitable positions for grafting key interaction residues of the ligand protein. These candidate residues were mutated to corresponding residues in the ligand protein at each relevant position and the mutated scaffold protein was co-minimized with receptor protein. Finally, the minimized complexes were evaluated by a scoring function deduced from statistical analysis of rigid binding data sets. As a test case, the binding epitope of barstar, the inhibitor of barnase, was grafted onto smaller proteins. Pheromone Er-1 (PDB entry 1erc) has been found to be a good scaffold. The calculated binding free energy for mutated Pheromone Er-1 is equivalent to that of barstar.

Algorithms↗
PubMed2000

Genetic network inference: from co-expression clustering to reverse engineering.

MOTIVATION: Advances in molecular biological, analytical and computational technologies are enabling us to systematically investigate the complex molecular processes underlying biological systems. In particular, using high-throughput gene expression assays, we are able to measure the output of the gene regulatory network. We aim here to review datamining and modeling approaches for conceptualizing and unraveling the functional relationships implicit in these datasets. Clustering of co-expression profiles allows us to infer shared regulatory inputs and functional pathways. We discuss various aspects of clustering, ranging from distance measures to clustering algorithms and multiple-cluster memberships. More advanced analysis aims to infer causal connections between genes directly, i.e. who is regulating whom and how. We discuss several approaches to the problem of reverse engineering of genetic networks, from discrete Boolean networks, to continuous linear and non-linear models. We conclude that the combination of predictive modeling with systematic experimental verification will be required to gain a deeper insight into living organisms, therapeutic targeting and bioengineering.

Animals↗
PubMed2000

Gene expression microarray data analysis for toxicology profiling.

When dealing with thousands of genes, all potentially interesting, it is desirable to rank the genes according to their degree of participation in a physiological process. Therefore, genes with the highest Shannon entropy and ERL can be selected as the best toxicity target candidates, permitting preclinical scientists to focus their research and resources on those genes.

Acetaminophen↗
PubMed2000

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

Animals↗