Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 829 records · Page 46Linked to original sources

PubMed1997

Repair effect of phenylpropanoid glycosides on thymine radical anion induced by pulse radiolysis.

Repair effects on thymine radical anion by six phenylpropanoid glycosides (PPGs), isolated from Pedicularis species, were studied using pulse radiolysis method. The thymine radical anion was produced by the reaction of hydrated electron with thymine. PPGs were added into the thymine solution saturated with N(2). Kinetic analysis showed that transient absorption spectrum of thymine radical anion formed at first, and then after several microseconds of pulse radiolysis changed to that of PPG radical anion. The evidence indicated that thymine radical anion was repaired through one-electron transfer between the radical anion and PPG. Electrophilic phenyl-substituted unsaturated carboxylic group containing in PPGs' structure was able to capture electron from thymine radical anion before it undergo reversible protonation. The reaction rate constants of electron transfer from thymine radical anion to PPGs were within 1.16-2.29 x 10(9) dm(3) mol(-1) s(-1).

Journal Article↗

PubMed1997

Protein-protein interaction specificity of Im9 for the endonuclease toxin colicin E9 defined by homologue-scanning mutagenesis.

The colicin DNase-specific immunity proteins interact with the endonuclease domain of the bacterial toxin colicin E9 with dissociation constants that span the millimolar to femtomolar affinity range. Among the non-cognate interactions Im2 shows the strongest binding toward the E9 DNase domain with a Kd of 10(-8) M, 6 orders of magnitude weaker than that of the cognate immunity protein Im9. Based on a NMR structure of Im9 that shows it to be a 4-helix protein, we have conducted a mutagenic scan in which elements of Im9 secondary structure were substituted into Im2 to precisely delineate regions that define specificity. Eleven chimeras were constructed, and their biological cross-reactivity toward colicins E2 and E9 was evaluated. From this set of mutants seven proteins were purified, and the Kd for their interaction with the E9 DNase domain was measured by a combination of stopped-flow fluorescence and subunit exchange kinetics. Our results show that immunity specificity is dominated by residues on helix II, accounting for 5 orders of magnitude binding specificity relative to Im2, and that packing interactions of helix II with its neighbor helix I and the loop connecting helix III with helix IV play minor roles. The conformational stability of these chimeric proteins was also determined. Proteins displaying an Im9 phenotype were all more stable than the parent Im2 protein, and surprisingly some chimeras were significantly more stable than either Im2 or Im9.

Amino Acid Sequence↗

PubMed1997

PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase.

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

3T3 Cells↗

PubMed1997

Evoked related potentials associated with and without an orienting reflex.

Late component event related potentials (ERP) and concomitant electrodermal activity (EDA) measures of the orienting reflex (OR) were undertaken in 50 normal subjects. Our group recently developed a model to quantify electrodermal activity in conventional ERP paradigms (auditory oddball) with short interstimulus intervals (ISI). The method was used to classify the presence or absence of skin conductance response following each auditory target stimulus. Using a conventional paradigm in which data is traditionally averaged, single-trial target ERPs were sorted into those with a skin conductance response OR (ERP[+OR]) and those without (ERP[-OR]) an OR, and ERP sub-averages of the two groups were derived. The ERP(+OR) showed significantly larger P300 amplitudes and relatively earlier N200 and P300 latency than those of the ERP(-OR). These findings suggest that using concomitant SCR-ERP measures, separate ERP related processes can be determined, that are complementary to the traditional average measure.

Adolescent↗

PubMed1997

PLC-gamma activation is required for PDGF-betaR-mediated mitogenesis and monocytic differentiation of myeloid progenitor cells.

To investigate the molecular mechanisms mediating hematopoietic cell differentiation and mitogenesis by activation of the platelet-derived growth factor beta receptor (PDGF-betaR), the wild type PDGF-betaR (PDGF-betaRWT) and tyrosine to phenylalanine mutants of the PDGF-betaR, including F751, F966, F970, F1009, F1021 and F1009/F1021 were overexpressed in FDC-P2 myeloid progenitor cells by retroviral-mediated gene transfer. Stimulation of PDGF-betaRWT and F966, F970 and F1009 infectants with PDGF-BB led to the increased expression of monocytic differentiation markers. In contrast, activation of PDGF-betaR in the parental line or the F1021 or F1009/F1021 mutant infectants failed to induce monocytic differentiation. PDGF-BB stimulation of PDGF-betaRWT, F751, F966, F970 and F1009 infectants led to pronounced DNA synthesis, whereas F1021 and F1009/F1021 infectants did not reveal any increase in mitogenesis when compared to that of the FDC-P2 line. While PDGF stimulation of FDC-P2 cells overexpressing PDGF-betaRWT led to a pronounced increase in inositol phosphate formation due to phospholipase C-gamma (PLC-gamma) activation, PDGF-BB induced phosphoinositol hydrolysis was completely abolished in the F1021 and F1009/F1021 infectants. GF 109203X, a specific inhibitor of protein kinase C (PKC) activation, fully blocked PDGF-betaR-mediated monocytic differentiation and mitogenesis. Taken together, these results suggest that stimulation of the PDGF-betaR signaling pathway can mediate monocytic differentiation when PDGF-betaR is expressed at sufficient levels and that activation of PLC-gamma and PKC plays a pivotal role in PDGF-betaR-mediated differentiation and mitogenesis in FDC-P2 cell system.

Binding Sites↗

PubMed1997

Actions of two highly potent organophosphorus neuropathy target esterase inhibitors in mammalian cell lines.

Neuropathy target esterase (NTE) is inhibited by many organophosphorus compounds that induce delayed neuropathy. This study examines two of the most potent NTE inhibitors, 2-octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (OBDPO) and ethyl octylphosphonofluoridate (EOPF), in cell lines with neural properties (PC-12 and NB41A3) and of nonneural origin (C6 and HeLa). NTE-like esteratic activity is higher in PC-12, HeLa and C6 cells than in NB41A3 cells and in each case is inhibited 50% by OBDPO and EOPF at 0.03-3.4 nM in vitro and by OBDPO at 0.080-36 nM in situ in culture. An NTE-like protein(s) of about 155 kDa is phosphorylated and labeled by [3H-octyl]OBDPO in these cell lines in the same order as their relative NTE esteratic activity. Cytotoxic levels of OBDPO and EOPF (300-500 microM) are generally 10(5) to > 10(7)-fold higher than required for NTE inhibition. PC-12 cells and OBDPO/[3H]OBDPO and EOPF are therefore suitable for research on non-lethal biochemical disruptions from NTE phosphorylation and aging.

Animals↗

PubMed1997

Human colorectal cancer (CRC) antigen CO17-1A/GA733 encoded by adenovirus inhibits growth of established CRC cells in mice.

The human colorectal carcinoma (CRC)-associated Ag CO17-1A/GA733, originally defined by mAbs CO17-1A and GA733, has been a useful target in passive immunotherapy of CRC patients with mAb and in active immunotherapy with anti-idiotypic Abs mimicking the CO17-1A or GA733 epitope. Both approaches have targeted single epitopes. We investigated the capacity of full-length CO17-1A/GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant adenovirus 5 (Ad5 GA733-2) to induce humoral, cellular, and/or protective immunity in mice. Ad5 GA733-2 induced Ag-specific Abs that reacted predominantly to CO17-1A- and GA733-unrelated epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with effector cells. Ad5 GA733-2-immune mice developed Ag-specific, proliferative lymphocytes of Th1 type and cytolytic lymphocytes. The use of Ad5 GA733-2 to immunize mice bearing established syngeneic CRC cells transfected with the human Ag induced significant and specific tumor regression. Cured mice resisted rechallenge with human CO17-1A/GA733 Ag-negative parental CRC cells, suggesting that targeting the human Ag on the murine transfectants induced protective immunity to other Ag expressed by the parental tumor. These results may explain the high potency of the recombinant vaccine. Thus, rAd5 GA733-2 may have potential as a vaccine for CRC patients.

Adenoviridae↗

PubMed1997

Aerosol particle transport and deaggregation phenomena in the mouth and throat.

The results of recent numerical simulations of aerosol particle transport and deaggregation phenomena in the mouth and throat are summarized. These results are used to comment upon the roles played by important factors influencing extrathoracic particle deposition and deaggregation phenomena. The results also provide a basis for comparing the performance of in vitro systems presently used for testing dry powder inhaler devices. Total deposition of aerosol particles in a 'physiologically faithful' three-dimensional (3D) reconstruction of the human mouth and throat closely mimics in vivo deposition data. On the other hand, aerosol particle deposition in a 3D 90 degrees -bend geometry does not accurately simulate aerosol filtration in the human mouth and throat. The numerically predicted regional deposition of aerosol particles closely matches that measured experimentally, in vitro, using the oropharyngeal cast from which the numerical reconstruction was made. A relatively weak dependence of particle deposition on flow-rate is observed with both 90 degrees -bend and 'real' oropharyngeal geometries, inconsistent with extrathoracic deposition in vivo. Two dimensional numerical simulations do not, in general, accurately simulate the conditions in the associated three-dimensional system. Quantitative differences between flow in the 90 degrees -bend and 'real' oropharyngeal geometries are especially significant in regards to the ability of the airstream to break apart particle agglomerates. For both geometries, it is observed that increasingly small particle agglomerates may be separated as the airflow rate increases from 30 to 200 lmin(-1). At flow-rates exceeding 60 lmin(-1), aggregated particles less than approximately 10-µm diameter may be separated by the airflow.

Journal Article↗

PubMed1997

Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin.

CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.

Anilino Naphthalenesulfonates↗

PubMed1997

Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction.

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.

Animals↗

PubMed1997

Nanoporous Molecular Sandwiches: Pillared Two-Dimensional Hydrogen-Bonded Networks with Adjustable Porosity

Crystal engineering of molecular materials is commonly frustrated by the absence of reliable structural paradigms that are needed for systematic design of crystal lattices with predictable structure and desirable function. This problem can be attributed, at least partially, to the absence of robust supramolecular motifs that serve as synthons for the assembly of crystal lattices. A novel class of molecular crystals based on two-dimensional hydrogen (H)-bonded networks comprising guanidinium ions and the sulfonate groups of alkane- or arenedisulfonate ions is described. The disulfonate ions act as pillars that connect opposing H-bonded sheets and form nanoporous galleries with one-dimensional channels. The flexibility of the H-bonded network allows the galleries to adapt to changes in the steric requirements of guest molecules that occupy the channels. This robustness reduces crystal engineering to the last remaining dimension, enabling rational adjustment of the gallery heights by choice of the disulfonate pillar.

Journal Article↗

PubMed1997

Fe- and Co-bleomycins bound to site specific and nonspecific DNA decamers: comparative binding and reactivity of their metal centers.

Co- and Fe-bleomycins (Blms) have been reacted with DNAa, d(GGAAGCTTCC)2, containing a specific site for cleavage, and DNAb, d(GGAAATTTCC)2, a closely related nonspecific 10-mer, to survey whether features of structure and reactivity of these adducts vary systematically as a function of the base sequence of the DNA oligomer. The ESR spectrum of NO-Fe(II)BlmDNAa is rhombically perturbed in comparison with that of NO-Fe(II)BlmDNAb, which is nearly identical to the spectrum of NO-Fe(II)Blm. The ESR spectrum of Fe(III)BlmDNAa in phosphate buffer is low-spin; that of Fe(III)BlmDNAb is high-spin as seen with Fe(III)Blm alone. According to absorbance spectroscopy, O2-Fe(II)BlmDNAa is stabilized in comparison with the DNAb adduct. Similar stabilization of O2-Co(II)Blm bound to DNAa but not to DNAb was also observed by ESR spectroscopy. HO2(-)-Co(III)Blm A2 binds in slow exchange on the NMR time scale to DNAa at its 5'-G-pyrimidine-3' site of cleavage. In contrast, fluorescence and NMR spectroscopy demonstrate that most of HO2(-)-Co(III)Blm A2 binds stoichiometrically in fast exchange to DNAb. The reactions of Fe(III)BlmDNAa and Fe(III)BlmDNAb with ascorbate and O2 reveal that the latter becomes activated and cleaves its 10-mer, producing base propenals, at a faster initial rate. Thus, in two series of metallobleomycins, (A) NO-Fe(II)Blm, O2-Fe(II)Blm, Fe(III)Blm in phosphate buffer, and HO2(-)-Fe(III)Blm and (B) O2-Co(II)Blm and HO2(-)-Co(III)Blm, the metal domain of each species interacts differently with DNA depending upon its base sequence.

Antibiotics, Antineoplastic↗

PubMed1997

Dosage compensation: X-repress yourself.

Dosage compensation in Caenorhabditis elegans involves the sex-specific recruitment to the X chromosome of a protein complex, the nature of which suggests that there are mechanistic links between chromosome segregation and global transcriptional regulation.

Animals↗

PubMed1997

Intramembrane substitutions in helix D of bacteriorhodopsin disrupt the purple membrane.

The Halobacterium salinarium purple membrane is a two-dimensional crystalline lattice containing bacteriorhodopsin (BR) and lipid. To test whether molecular packing within the lipid bilayer influences the structural stability of the lattice, BR mutants substituted on the membrane-embedded surface of the protein were expressed in H. salinarium. Lattice stability was assessed by equilibrium density centrifugation of cell lysates containing similar amounts of BR. BR was distributed in low (1.12 to 1.15 g/ml) and high density (1.18 g/ml) membrane fractions. The high density fraction comprised 89% of the total BR in wild-type, but only 19% (G113L), 29% (I117A), 52% (G116L) and 79% (I117F) in the mutants. In each case, this fraction contained BR in a lattice form: its absorption maximum was blue-shifted by < or = 4 nm relative to the wild-type lattice, its light-dark difference spectrum was normal, and its circular dichroism spectrum retained a bilobed feature characteristic of the lattice. Thus, the substitutions do not significantly alter the tertiary structure of the protein. In the low density fraction, the absorption maximum of BR was blue-shifted by 2 to 4 nm relative to the corresponding high density fraction, and the bilobed circular dichroism feature was attenuated (I117F and G116L) or absent (G113L and I117A). This suggests that the substitutions disrupt lattice stability, causing an accumulation of BR monomers or small aggregates. These results support a model in which the BR lattice is stabilized by hydrophobic packing at specific protein-protein and protein-lipid interfaces within the membrane bilayer.

Bacteriorhodopsins↗

PubMed1997

Requirement of phosphatidylinositol-3 kinase for activation of JNK/SAPKs by PDGF.

The molecular mechanism by which cell surface receptors stimulate the serine/threonine kinase activity of c-Jun N-terminal kinases (JNKs) was investigated using a transient cotransfection experiments in COS-7 cells. Our data demonstrate that JNK activity is potently induced by platelet derived growth factor (PDGF) upon expression of beta PDGFR wild type (beta RWT). However, PDGF failed to mediate JNK activation in cells expressing beta PDGFR mutant lacking the binding site for phosphatidylinositol-3 (PI-3) kinase but not for phospholipase C gamma (PLC gamma) or Syp. Consistent with this result, a PI-3 kinase inhibitor, wortmannin inhibited activation of JNK by PDGF. Furthermore, overexpression of P110 the catalytic domain of PI-3 kinase was sufficient for activation of JNKs which could be efficiently inhibited by dominant negative forms of Ras, Rac but not of RhoA or Cdc42. Taken together all of these findings suggest that activation of JNK by PDGF involves receptor association with PI-3 kinase activity, which in turn acts on a ras- and rac-dependent pathway.

Androstadienes↗

PubMed1997

Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury.

To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

Animals↗

PubMed1997

Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10.

Streptomyces viridifaciens MG456-hF10 produces the antitumor agent valanimycin, which is a member of a family of antibiotics containing the azoxy group. An enzyme involved in the biosynthesis of valanimycin has been purified 360-fold from S. viridifaciens. This enzyme, isobutylamine N-hydroxylase, catalyzes the oxidation of isobutylamine to isobutylhydroxylamine in the presence of oxygen and a reduced flavin cofactor. Unlike other known N-hydroxylases, isobutylamine N-hydroxylase cannot carry out the reduction of the flavin cofactor. Rather, the reduced flavin is supplied by a separate flavin reductase that is present in extracts of S. viridifaciens. The reduced flavin cofactor could also be supplied by the flavin mononucleotide reductase of Vibrio fischeri. The requirement for molecular oxygen and a reduced flavin indicates that the N-hydroxylase is a flavin monooxygenase and that the mechanism for the hydroxylation is likely to proceed via the formation of a flavin 4a-hydroperoxide. Isobutylamine N-hydroxylase exhibited a subunit molecular mass of 40 kDa and existed in dimeric or trimeric form depending upon buffer conditions. The pI of the protein was found to be ca. 5.1 and the enzyme exhibited a sensitivity to thiol-directed reagents.

Amino Acid Sequence↗

PubMed1997

The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz.

Homeobox genes specify cell fate and positional identity in embryos throughout the animal kingdom. Paradoxically, although each has a specific function in vivo, the in vitro DNA-binding specificities of homeodomain proteins are overlapping and relatively weak. A current model is that homeodomain proteins interact with cofactors that increase specificity in vivo. Here we use a native binding site for the homeodomain protein Fushi tarazu (Ftz) to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone bind only high-affinity sites. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects. This phenotype is a result of abnormal ftz function because it is expressed but fails to activate downstream target genes. Cooperative interaction between homeodomain proteins and cofactors of different classes may serve as a general mechanism to increase HOX protein specificity and to broaden the range of target sites they regulate.

Animals↗