Biomedical subjects

W Li

Publications and source records attributed to W Li.

At least 163 records · Page 9Linked to original sources

PubMed2001

Synthesis of substituted 5[H]phenanthridin-6-ones as potent poly(ADP-ribose)polymerase-1 (PARP1) inhibitors.

1-, 2-, 3-, 4-, 8-, or 10-Substituted 5(H)phenanthridin-6-ones were synthesized and found to be potent PARP1 inhibitors. Among the 28 compounds prepared, some showed not only low IC(50) values (compound 1b, 10 nM) but also desirable water solubility characteristics. These properties, which are superior to the common PARP1 inhibitors such as benzamides and isoquinolin-1-ones, are essential for potential therapeutic usage. The variety of compounds allows SAR analysis of favored substituents and substituted positions on 5(H)phenanthridin-6-one ring.

Enzyme Inhibitors↗

PubMed2001

Antioxidants prevent depletion of [Mg2+]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: possible relationship to alcohol-induced stroke.

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis, and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Mg(2+) ([Mg(2+)](i)) depletion associated with cerebral vascular injury induced by alcohol. Exposure of cultured canine cerebral vascular smooth muscle cells to alcohol (10-100 mM) for 24 h induced marked depletion in [Mg(2+)](i) (i.e., approximately 30-65%, depending upon alcohol concentration). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Mg(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the depletion of [Mg(2+)](i) induced by exposure to 10-100 mM ethanol. These results indicate that alpha-tocopherol and PDTC prevent decreases in [Mg(2+)](i) produced by ethanol. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in prevention and amelioration of cerebral vascular injury and stroke in alcoholics.

Alcoholism↗

PubMed2001

Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity.

Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, we examined 10 ovarian cancer cell lines for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, we found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because we did not detect dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation. Lack of APAF-1 activity correlated in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers.

Apoptosis↗

PubMed2001

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).

Binding Sites↗

PubMed2001

Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress.

Diabetes mellitus (DM) is a primary risk factor for cardiovascular disease. Although recent studies have demonstrated an important role for extracellular matrix metalloproteinases (MMPs) in atherosclerosis, little is known about the effects of hyperglycemia on MMP regulation in vascular cells. Gelatin zymography and Western blot analysis revealed that the activity and expression of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, were significantly increased in vascular tissue and plasma of two distinct rodent models of DM. Bovine aortic endothelial cells (BAECs) grown in culture did not express MMP-9 constitutively; however, chronic (2-week) incubation with high glucose medium induced MMP-9 promoter activity, mRNA and protein expression, and gelatinase activity in BAECs. On the other hand, high glucose culture did not change MMP-9 activity from vascular smooth muscle cells or macrophages. Electron paramagnetic resonance studies indicate that BAECs chronically grown in high glucose conditions produce 70% more ROS than do control cells. Enhanced MMP-9 activity was significantly reduced by treatment with the antioxidants polyethylene glycol-superoxide dismutase and N-acetyl-L-cysteine but not by inhibitors of protein kinase C. In conclusion, vascular MMP-9 activity is increased in DM, in part because of enhanced elaboration from vascular endothelial cells, and oxidative stress plays an important role. This novel mechanism of redox-sensitive MMP-9 expression by hyperglycemia may provide a rationale for antioxidant therapy to modulate diabetic vascular complications.

Animals↗

PubMed2001

Identification and characterization of a melanin-concentrating hormone receptor.

Melanin-concentrating hormone (MCH), a neuropeptide expressed in central and peripheral nervous systems, plays an important role in the control of feeding behaviors and energy metabolism. An orphan G protein-coupled receptor (SLC-1/GPR24) has recently been identified as a receptor for MCH (MCHR1). We report here the identification and characterization of a G protein-coupled receptor as the MCH receptor subtype 2 (MCHR2). MCHR2 has higher protein sequence homology to MCHR1 than any other G protein-coupled receptor. The expression of MCHR2 has been detected in many regions of the brain. In contrast to MCHR1, which is intronless in the coding region and is located at the chromosomal locus 22q13.3, the MCHR2 gene has multiple exons and is mapped to locus 6q21. MCHR2 is specifically activated by nanomolar concentrations of MCH, binds to MCH with high affinity, and signals through Gq protein. This discovery is important for a full understanding of MCH biology and the development of potential therapeutics for diseases involving MCH, including obesity.

Alternative Splicing↗

PubMed2001

Nanocrystalline metal oxides as unique chemical reagents/sorbents.

A new family of porous inorganic solids based on nanocrystalline metal oxides is discussed. These materials, made up of 4-7 nm MgO, CaO, Al2O3, ZnO, and others, exhibit unparalleled destructive adsorption properties for acid gases, polar organics, and even chemical/biological warfare agents. These unique sorption properties are due to nanocrystal shape, polar surfaces, and high surface areas. Free-flowing powders or consolidated pellets are effective, and pore structure can be controlled by consolidation pressures. Chemical properties can be adjusted by choice of metal oxide as well as by incorporating other oxides as monolayer films.

Journal Article↗

PubMed2001

New stopping criteria for segmenting DNA sequences.

We propose a solution on the stopping criterion in segmenting inhomogeneous DNA sequences with complex statistical patterns. This new stopping criterion is based on Bayesian information criterion in the model selection framework. When this criterion is applied to telomere of S. cerevisiae and the complete sequence of E. coli, borders of biologically meaningful units were identified, and a more reasonable number of domains was obtained. We also introduce a measure called segmentation strength which can be used to control the delineation of large domains. The relationship between the average domain size and the threshold of segmentation strength is determined for several genome sequences.

Bayes Theorem↗

PubMed2001

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis.

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 microM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H(2)O(2)) exposure (100 microM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H(2)O(2) preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H(2)O(2) stimulated divergent pathways, with caspases being activated only by H(2)O(2). The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H(2)O(2), suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H(2)O(2). The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses.

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.

Animals↗

PubMed2001

Eukaryotic initiation factors 4A (eIF4A) and 4G (eIF4G) mutually interact in a 1:1 ratio in vivo.

mRNA translation in eukaryotic cells involves a set of proteins termed translation initiation factors (eIFs), several of which are involved in the binding of ribosomes to mRNA. These include eIF4G, a modular scaffolding protein, and eIF4A, an RNA helicase, of which two closely related forms are known in mammals, eIF4A(I) and eIF4A(II). In mammals, eIF4G possesses two independent sites for binding eIF4A, whereas in other eukaryotes (e.g. yeast) only one site appears to be present, thus raising the issue of the stoichiometry of eIF4G.eIF4A complexes in different eukaryotes. We show that in human embryonic kidney cells eIF4G is associated with eIF4A(I) or eIF4A(II) but not with both simultaneously, suggesting a stoichiometry of 1:1 rather than 1:2. To confirm this, eIF4A(I) or eIF4A(II) was expressed in a tagged form in these cells, and complexes with eIF4G were again isolated. Complexes containing tagged eIF4A(I) or eIF4A(II) contained no endogenous eIF4A, supporting the notion that eIF4G binds only one molecule of eIF4A. Each binding site in eIF4G can bind either eIF4A(I) or eIF4A(II). The data imply that the second binding site in mammalian eIF4A does not bind an additional eIF4A molecule and that initiation factor complexes in different eukaryotes contain one eIF4A per eIF4G.

Amino Acid Sequence↗

PubMed2001

Abrogation of disease development in plants expressing animal antiapoptotic genes.

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant-pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host-cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant-pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Apoptosis↗

PubMed2001

An efficient synthesis of the taxane-derived anticancer agent ABT-271.

ABT-271, 1, has been identified as a promising anticancer agent. ABT-271 is a novel taxane possessing a C9-(R)-hydroxyl group as opposed to a C9-ketone which is present in Taxol and Taxotere. To further evaluate ABT-271 as a potential anticancer agent, an efficient synthesis was developed which allows the large scale synthesis of ABT-271. Ketalization of the 7,9-diol of 9-DHAB-III, 2, allows selective removal of the C13-acetate with phenyllithium. The resulting C13-hydroxyl group is then acylated using LiHMDS and beta-lactam 22 to give ABT-271 in protected form. The protecting groups were removed first by acidic hydrolysis followed by basic hydrolysis to provide ABT-271. Application of this synthetic sequence provided over 600 g of ABT-271, 1.

Antineoplastic Agents↗

PubMed2001

Interleukin-6 protects against Fas-mediated death by establishing a critical level of anti-apoptotic hepatic proteins FLIP, Bcl-2, and Bcl-xL.

Previous studies showed that following acute carbon tetrachloride (CCl(4)) treatment, interleukin-6 null (IL-6-/-) mice develop increased hepatocellular injury, defective regeneration, delayed wound healing, and increased hepatocyte apoptosis. Pretreatment with IL-6 prior to CCl(4) reduces injury, hepatocyte apoptosis, and accelerates regeneration in both IL-6-/- and +/+ livers. To demonstrate whether IL-6 can prevent liver injury that involves direct stimulation of hepatocyte apoptosis, IL-6-/- and +/+ mice were treated with the Fas agonist, Jo-2 mAb. At low Fas agonist doses, IL-6+/+ mice developed mild hepatic injury and survived, whereas IL-6-/- mice developed severe apoptotic hepatitis within 12 h and died. Pretreatment with IL-6 improved survival in IL-6-/- mice and reduced injury in both IL-6-/- and +/+ livers. The direct anti-apoptotic effects of IL-6 were demonstrated in vitro as IL-6 decreased Fas-mediated apoptosis in both IL-6-/- and +/+ primary hepatocyte cultures, and suggested that IL-6-/- hepatocytes have a pre-existing defect in anti-apoptotic pathways. After Fas activation, IL-6-/- livers demonstrated evidence of both proximal and distal alterations in the apoptotic pathways including elevated caspase 8 and 3 activation-associated fragments, and loss of cytochrome c staining. IL-6-/- livers had reduced pre-existing protein expression of the anti-apoptotic factors Bcl-2 and Bcl-xL as well as more rapid degradation of FLIP following Fas treatment that appeared to be post-transcriptionally regulated. FLIP is a crucial proximal inhibitor of caspase 8 activation in Fas, tumor necrosis factor, and DR3/DR4-mediated apoptosis, and Bcl-2 and Bcl-xL more downstream anti-apoptotic regulators. IL-6 may function as a critical anti-apoptotic factor in the liver by its ability to establish and maintain an adequate level of FLIP and downstream anti-apoptotic factors.

Animals↗

PubMed2001

Differential requirement for Rho family GTPases in an oncogenic insulin-like growth factor-I receptor-induced cell transformation.

Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells. MAPK, phospholipase C gamma, and Stat3 were activated to the same extent by NM1 and its two mutants, suggesting that activation of these pathways, individually or in combination, was not sufficient for NM1-induced cell transformation. The mutant dS2 has decreased IRS-1 phosphorylation levels and IRS-1-associated phosphatidylinositol 3'-kinase activity, suggesting that this impairment may be in part responsible for the defectiveness of dS2. We show that Rho family members, RhoA, Rac1, and Cdc42 are activated by NM1, and this activation, particularly RhoA and Cdc42, is attenuated in both mutants of NM1. Dominant negative mutants of Rho, Rac, and Cdc42 inhibited NM1-induced cell transformation, as measured by focus and colony forming ability. Dominant negative Rho most potently inhibited the focus forming activity, whereas Cdc42 was most effective in inhibiting the colony forming ability of NM1-expressing cells. Conversely, constitutively activated (ca) Rho is more effective than ca Rac or ca Cdc42 in rescuing the focus forming ability of the mutants. By contrast, ca Cdc42 is most effective in rescuing the colony forming ability of both mutants.

3T3 Cells↗

PubMed2001

Analysis of HIV-1 reverse transcriptase and protease sequences in paired plasma and lymphoid tissue specimens from HIV-1 infected individuals.

OBJECTIVES: To determine how representative the genotype of HIV-1 circulating in plasma is of the genotype of the virus present in lymphoid tissue. METHODS: Paired plasma and tonsillar tissue samples were prospectively obtained from patients with various levels of plasma HIV-1 RNA who were receiving combination antiretroviral therapy. HIV-1 reverse transcriptase and protease sequences were amplified from plasma and lymphoid tissue specimens by nested polymerase chain reaction and analyzed using an automated sequencing system. Results were compared with consensus HIV-1 sequences to determine whether drug-resistance mutations were present in the regions analyzed. RESULTS: HIV-1 protease sequences were compared in 11 plasma/tissue pairs obtained from eight patients; HIV reverse transcriptase sequences were compared in 12 plasma/tissue pairs obtained from nine patients. Sequence homology between plasma and tissue RNA, tissue RNA and DNA, and plasma and tissue DNA ranged from 97% to 100%. Few discrepancies were found when the percentage of mutant sequences at resistance codons was compared among paired samples. In most instances, tissue RNA or plasma contained a higher percentage of mutant sequences than did tissue DNA. CONCLUSION: The genotype of plasma HIV-1 is similar to the genotype of the virus in lymphoid tissue. Resistance studies using plasma samples should provide accurate information regarding the genotype of HIV-1 in lymphoid tissues.

HIV Infections↗

PubMed2001

Il-12 antagonism enhances apoptotic death of T cells within hepatic allografts from Flt3 ligand-treated donors and promotes graft acceptance.

Mouse livers are accepted across MHC barriers and induce donor-specific tolerance without immunosuppressive therapy. By contrast, livers from donors treated with Flt3 ligand, which dramatically increases hepatic interstitial dendritic cells, are rejected acutely (median survival time 5 days). This switch from tolerance to rejection is associated with a marked reduction in apoptotic activity of graft-infiltrating cells. We hypothesized that IL-12 production by enhanced numbers of donor APC might inhibit apoptosis, promote expansion of Th1 cells, and play a key role in liver rejection. Therefore, C3H (H2(k)) recipients of liver grafts from Flt3 ligand-treated B10 donors were given neutralizing anti-IL-12 mAb (200 or 500 microg) on days 0 and 2 after transplant. Graft survival was markedly prolonged at the higher mAb dose, with 50% of grafts surviving >100 days. This effect was associated with reductions in IFN-gamma gene transcripts within the graft-infiltrating cell population and with reductions in circulating IFN-gamma and IL-10 levels, donor-specific CTL and NK cell activities, and circulating alloantibody levels. At the same time, there were marked increases in apoptotic (TUNEL(+)) CD4(+) and especially CD8(+) cells, both within the grafts and in spleens of anti-IL-12 mAb-treated mice. In vitro, exogenous IL-12 inhibited apoptotic death induced in naive allogeneic T cells by liver nonparenchymal cells. These findings suggest that suppression of rejection by IL-12 antagonism, linked to restoration of apoptotic activity within the peripheral alloreactive T cell population, is important for liver allograft survival and tolerance induction.

Adjuvants, Immunologic↗

PubMed2001

Characterization of insulin-like growth factor I (IGF-I) receptor mutants for their effects on IGF-I- and interleukin 4-mediated DNA synthesis of 32D cells.

Recently we demonstrated that overexpression of the wild type insulin-like growth factor I receptor (IGF-IRWT) in 32D myeloid progenitor cells led to cell proliferation in response to interleukin 4 (IL-4) as well as insulin-like growth factor I (IGF-I) in the absence of insulin receptor substrate expression (Soon, L., Flechner, L., Gutkind, J. S., Wang, L. H., Baserga, R., Pierce, J. H., and Li, W. (1999) Mol. Cell. Biol. 19, 3816-3828). To understand the structural importance of insulin-like growth factor I receptor (IGF-IR) in mediating IL-4- and IGF-I-induced DNA synthesis, we transfected various mutants of IGF-IR to 32D cells. Our results show that most mutants, including Y1250F, Y1251F, Y1250F/Y1251F, S1280A/S1281A/S1282A/S1283A, Y1316F, and 1245d, still retained mitogenic response toward IGF-I or IL-4. However, the Y950F, Y1131F, and Y1135F mutants were not able to respond to either ligand. The H1293F/K1294R and 1293d mutants reduced response toward IGF-I but not to IL-4. Phosphorylation of Shc was greatly reduced in those three mutants that lost mitogenic response. The MAPK activity was much lower in Y1131F and Y1135F mutants, indicating the importance of the Shc/MAPK pathway in IGF-I-induced mitogenesis. Importantly, the synergistic effect of these two factors on DNA synthesis was not affected in cells expressing most of the mutants, even in those three that had lower mitogenic response toward a single ligand. These results suggest that an unidentified pathway(s) may be induced upon co-addition of IGF-I and IL-4 that sustains the intact mitogenesis.

Adaptor Proteins, Signal Transducing↗