Biomedical subjects

S Liang

Publications and source records attributed to S Liang.

At least 19 recordsLinked to original sources

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↗

PubMed1999

Age-related variation in the interstitial tissues of the cardiac conduction system; and autopsy study of 230 Han Chinese.

The amount of fatty and fibrous tissues in 230 Han Chinese who died of noncardio-vascular diseases has been studied by a semi-quantitative method and analysed by chi-square test. The results have shown some consistency. Generally, in the sino-atrial node (SAN), fibrosis and fatty influtratin appear only after 40 years of age and increase one grade with every 20 years. The atrio-ventricular node (AVN) showed fatty change after 30 years of age and fibrosis appeared after 60. In the His bundle (HB), fatty infiltration and fibrosis appear after 40 years. The left bundle branch (LBB) showed similar changes. The appearance of fibrosis in the AVN seems to be later than that reported by Lev.

Adolescent↗

PubMed1999

Emerging HIV infections with distinct subtypes of HIV-1 infection among injection drug users from geographically separate locations in Guangxi Province, China.

Heroin users from Guangxi province, a southern province of China that borders Vietnam in the south and Yunnan province in China in the west, were studied for prevalence and risk factors for HIV-1 infection. Viral env sequences from HIV-1-positive individuals were also determined for subtypes of HIV-1. The overall HIV prevalence among 227 heroin users was 40%. Most had used drugs for < or = 3 years. Sharing of injection equipment and unprotected sex were significantly associated with HIV-1 infection. Subtypes C and E HIV-1 were detected in infected heroin users and were sharply segregated in two geographic locations: only subtype C was found in a border city with Yunnan province, whereas only subtype E was found in a city bordering northern Vietnam. HIV-1 strains within each subtype were remarkably homogenous, with a mean intersubject DNA distance of 2.32% for subtype E and 1.13% for subtype C, respectively. Phylogenetic analysis of C2-V5 region of Guangxi subtype E env sequences revealed significant clustering with subtype E sequences from southern Vietnam and Cambodia. These results suggest that HIV-1 infection among heroin users in Guangxi represents two emerging epidemics initiated from distinct sources: one from Vietnam and another from Yunnan province. Factors associated with HIV-1 infection were not restricted to injection practices. Unprotected sexual behaviors are likely to increase the probability of HIV transmission beyond this high-risk population. Designing and implementing effective intervention strategies targeted toward both injection drug use and high risk sexual behavior are urgently needed to further reduce HIV-1 spread in China.

China↗

PubMed1999

Activities of constitutive promoters in Escherichia coli.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains.

Cell Division↗

PubMed1999

Expression and localisation of dynamin and syntaxin during neural development and neuromuscular synapse formation.

The expression and subcellular localisation of dynamin and syntaxin were examined during the periods of motor neuron development and neuromuscular synaptogenesis in the mouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinal cord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent ages examined. Reverse transcription and polymerase chain reaction (RT-PCR) identified low levels of all three carboxy-terminal splicing forms of dynamin I in spinal cord from as early as E10. During the period of maturation of spinal neurons, from E10 to the first postnatal day (P0), the short carboxy-terminal splicing form of dynamin I (dynamin I*b) was up-regulated, as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining became colocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hours of the commencement of synapse formation and throughout subsequent development. In contrast, dynamin, which is important for activity-dependent synaptic vesicle recycling and, thus, sustained neurotransmission, could not be detected at most newly formed synapses until several days after synapse formation. The delayed appearance of dynamin at the synapse, thus, heralds the neonatal development of robust synaptic transmission at the neuromuscular junction.

Animals↗

PubMed1999

Solution structure of the major alpha-amylase inhibitor of the crop plant amaranth.

alpha-Amylase inhibitor (AAI), a 32-residue miniprotein from the Mexican crop plant amaranth (Amaranthus hypochondriacus), is the smallest known alpha-amylase inhibitor and is specific for insect alpha-amylases (Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R., and Pongor, S. (1994) J. Biol. Chem. 269, 23675-23680). Its disulfide topology was confirmed by Edman degradation, and its three-dimensional solution structure was determined by two-dimensional 1H NMR spectroscopy at 500 MHz. Structural constraints (consisting of 348 nuclear Overhauser effect interproton distances, 8 backbone dihedral constraints, and 9 disulfide distance constraints) were used as an input to the X-PLOR program for simulated annealing and energy minimization calculations. The final set of 10 structures had a mean pairwise root mean square deviation of 0.32 A for the backbone atoms and 1.04 A for all heavy atoms. The structure of AAI consists of a short triple-stranded beta-sheet stabilized by three disulfide bonds, forming a typical knottin or inhibitor cystine knot fold found in miniproteins, which binds various macromolecular ligands. When the first intercystine segment of AAI (sequence IPKWNR) was inserted into a homologous position of the spider toxin Huwentoxin I, the resulting chimera showed a significant inhibitory activity, suggesting that this segment takes part in enzyme binding.

Amino Acid Sequence↗

PubMed1999

Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena.

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis. To map the disulfide bonds, native SHL-I was proteolytically digested. The resulting peptides were separated by reverse phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis indicated the presence of one disulfide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2-carboxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performance liquid chromatography. Four M Guanidine-HCl was found to increase the yields of partially reduced peptides prominently. The free thiols were carboxamidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of the partially reduced and alkylated SHL-I with that of the intact peptide. Finally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys14, Cys7-Cys19, Cys13-Cys26.

Amino Acid Sequence↗

PubMed1999

Three-dimensional structure of Selenocosmia huwena lectin-I (SHL-I) from the venom of the spider Selenocosmia huwena by 2D-NMR.

The three-dimensional structure of native SHL-I, a lectin from the venom of the Chinese bird spider Selenocosmia huwena, has been determined from two-dimensional 1H NMR spectroscopy recorded at 500 and 600 MHz. The best 10 structures have NOE violation <0.3 A, dihedral violation <2 deg, and average root-mean-square differences of 0.85 + 0.06 A over backbone atoms. The structure consists of a three-stranded antiparallel beta-sheet and three turns. The three disulfide bridges and three-stranded antiparallel beta-sheet form a inhibitor cystine knot motif which is adopted by several other small proteins, such as huwentoxin-I, omega-conotoxin, and gurmarin. The C-terminal fragment from Leu28 to Trp32 adopts two sets of conformations corresponding to the cis and trans conformations of Pro31. The structure of SHL-I also has high similarity with that of the N-terminus of hevein, a lectin from rubber-tree latex.

Amino Acid Sequence↗