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Biomedical subjects

M Li

Publications and source records attributed to M Li.

At least 361 records · Page 20Linked to original sources
PubMed2000

mu-Oxo-bis[(2,3,12,13-tetrabromo-5,10,15, 20-tetraphenylporphyrinato)iron(III)] bis(dichloromethane) solvate.

The precise structure of the title compound, [Fe(2)O(C(44)H(24)Br(4)N(4))(2)].2CH(2)Cl(2), is reported. The Fe-N distances are non-equivalent in pairs because of the asymmetric peripheral substitution; the values are 2.098 A to the brominated rings and 2.041 A to the other two rings. The Fe-O bond distance is 1.7583 (4) A. The molecule has required twofold symmetry so that there is one unique porphyrin macrocycle and one Fe-O bond length in contrast to a previous report on the same species.

Crystallography, X-Ray↗
PubMed2000

Molecular-replacement studies of Trichosanthes kirilowii lectin 1: a structure belonging to the family of type 2 ribosome-inactivating proteins.

Trichosanthes kirilowii lectin 1 (TKL-1) isolated from the tuber of T. kirilowii consists of two chains, each with a molecular weight of about 30 kDa. It has immunological properties which are similar to some ribosome-inactivating proteins (RIPs). TKL-1 was crystallized in space group P2(1)2(1)2(1) and diffraction data were collected to 2.7 A resolution. The molecular-replacement method was applied to solve the structure, using different chains of ricin, abrin-a and trichosanthin as search models. A set of consistent solutions was further verified by R(omit) profile analysis. In addition, the spatial arrangement of the two chains of TKL-1 is identical to that of type 2 RIPs.

Crystallization↗
PubMed2000

Crystallization and preliminary X-ray diffraction studies of human epidermal growth factor.

Human epidermal growth factor (hEGF), a 6.2 kDa protein of 53 amino acids with three internal disulfide bridges, has been crystallized by the hanging-drop method. hEGF crystallizes in space group P3(1)21 (or P3(2)21) using MgCl(2) as precipitant, with unit-cell parameters a = b = 61.4, c = 87.0 A. Another type of crystal, obtained using NaCl as precipitant, belongs to a tetragonal point group and has unit-cell dimensions a = b = 102.5, c = 166.6 A. The trigonal crystals with the smaller unit cell diffract X-rays better and a native data set from a single crystal has been collected to 3.0 A resolution.

Crystallization↗
PubMed2000

A study on ultrasonic solid horns for flexural mode

The transfer matrix method is introduced into the analysis of ultrasonic flexural solid horns. The frequency and magnification equations of a horn are derived by using the transfer matrix method. Analyzed are the characteristics of three types of circular cross-section horns: exponent, cone, and catenary. The results are partly verified experimently.

Journal Article↗
PubMed2000

Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

Adenylyl Cyclases↗
PubMed2000

Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells.

Induction of apoptotic cell death generally requires the participation of cysteine proteases belonging to the caspase family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Surprisingly, TNF-alpha treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was significantly reduced in cells infected with a vaccinia virus mutant lacking the caspase inhibitor B13R. Furthermore, TNF-alpha also induced necrotic-like cell death of fibroblasts in the presence of peptidyl caspase inhibitors. In both cases, necrosis was accompanied by generation of superoxide species. Caspase inhibitors also sensitized fibroblasts to killing by double-stranded RNA and gamma interferon. In all cases, cell death was efficiently blocked by antioxidants or mitochondrial respiratory chain inhibitors. These results define a new mitochondrion-dependent mechanism which may be important in the killing of cells infected with viruses encoding caspase inhibitors.

Animals↗
PubMed2000

Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon.

The alpha/beta interferon (IFN-alpha/beta) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-alpha/beta-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-alpha/beta genes, including IFN-beta. It is known that transactivation of the IFN-beta promoter depends on NF-kappaB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-kappaB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-kappaB pathway during delNS1 virus infection prevented the transactivation of the IFN-beta promoter, demonstrating a functional link between NF-kappaB activation and IFN-alpha/beta synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-kappaB pathway and of IFN-beta synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-kappaB pathway may thus play a key role in the pathogenesis of influenza A virus.

Animals↗
PubMed2000

Peroxisome proliferator-activated receptor gamma-dependent repression of the inducible nitric oxide synthase gene.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that activates target gene transcription in a ligand-dependent manner. In addition, liganded PPARgamma can inhibit transcription of genes induced by gamma interferon (IFN-gamma) and/or lipopolysaccharides (LPSs), including the inducible nitric oxide synthase (iNOS) gene. Inhibition of the iNOS promoter is achieved partially through antagonizing the activities of NF-kappaB, AP-1, and STAT1, which are known to mediate effects of LPS and IFN-gamma. Previous studies have suggested that transrepression of these factors by nuclear receptors involves competition for limiting amounts of the general coactivators CREB-binding protein (CBP) and p300. CBP and p300 are thought to be recruited to nuclear receptors through bridging factors that include SRC-1, although CBP also interacts directly with PPARgamma through its amino terminus. These observations have raised questions concerning the involvement of SRC-1-like factors in CBP recruitment and transrepression. We here provide evidence that PPARgamma's ability to repress iNOS transcription requires the ligand-dependent charge clamp that mediates interactions with CBP and SRC-1. Single amino acid mutations in PPARgamma that abolished ligand-dependent interactions with SRC-1 and CBP not only resulted in complete loss of transactivation activity but also abolished transrepression. Conversely, a CBP deletion mutant containing the SRC-1 interaction domain but lacking the N-terminal PPARgamma interaction domain was inactive as a PPARgamma coactivator and failed to rescue transrepression. Together, these findings are consistent with a model in which transrepression by PPARgamma is achieved by targeting CBP through direct interaction with its N-terminal domain and via SRC-1-like bridge factors.

Animals↗
PubMed2000

ATF6 as a transcription activator of the endoplasmic reticulum stress element: thapsigargin stress-induced changes and synergistic interactions with NF-Y and YY1.

ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucose-regulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane domain, is primarily associated with the perinuclear region. Upon Tg stress, the ATF6 protein level dropped initially but quickly recovered with the additional appearance of a faster-migrating form. This new form of ATF6 was recovered as soluble nuclear protein by biochemical fractionation, correlating with enhanced nuclear localization of ATF6 as revealed by immunofluorescence. Optimal ATF6 stimulation requires at least two copies of the ERSE and the integrity of the tripartite structure of the ERSE. Of primary importance is a functional NF-Y complex and a high-affinity NF-Y binding site that confers selectivity among different ERSEs for ATF6 inducibility. In addition, we showed that YY1 interacts with ATF6 and in Tg-treated cells can enhance ATF6 activity. The ERSE stimulatory activity of ATF6 exhibits properties distinct from those of human Ire1p, an upstream regulator of the mammalian unfolded protein response. The requirement for a high-affinity NF-Y site for ATF6 but not human Ire1p activity suggests that they stimulate the ERSE through diverse pathways.

3T3 Cells↗
PubMed2000

Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcomas, body cavity-based lymphomas, and some forms of Castleman's disease. The K9 open reading frame of KSHV encodes a viral interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction and as an oncogene to induce cell growth transformation. We demonstrate that KSHV vIRF directly interacts with cellular transcriptional coactivator p300 and displaces p300/CBP-associated factor from p300 complexes. This interaction inhibits the histone acetyltransferase activity of p300, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure. As a consequence, vIRF expression markedly alters cellular cytokine expression, which is regulated by acetylation of nucleosomal histones. These results demonstrate that KSHV vIRF interacts with and inhibits the p300 transcriptional coactivator to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression. These studies also illustrate how a cellular gene captured by a herpesvirus has evolved several functions that suit the needs of the virus.

3T3 Cells↗
PubMed2000

Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3beta.

Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3beta (GSK-3beta). The increased phosphorylation of GSK-3beta by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3beta in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3beta mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3beta and inhibits its apoptotic activity in neurons.

Animals↗
PubMed2000

Relative contributions of "pressure pump" and "peristaltic pump" to gastric emptying.

The relative contributions to gastric emptying from common cavity antroduodenal pressure difference ("pressure pump") vs. propagating high-pressure waves in the distal antrum ("peristaltic pump") were analyzed in humans by high-resolution manometry concurrently with time-resolved three-dimensional magnetic resonance imaging during intraduodenal nutrient infusion at 2 kcal/min. Gastric volume, space-time pressure, and contraction wave histories in the antropyloroduodenal region were measured in seven healthy subjects. The subjects fell into two distinct groups with an order of magnitude difference in levels of antral pressure activity. However, there was no significant difference in average rate of gastric emptying between the two groups. Antral pressure history was separated into "propagating high-pressure events" (HPE), "nonpropagating HPEs," and "quiescent periods." Quiescent periods dominated, and average pressure during quiescent periods remained unchanged with decreasing gastric volume, suggesting that common cavity pressure levels were maintained by increasing wall muscle tone with decreasing volume. When propagating HPEs moved to within 2-3 cm of the pylorus, pyloric resistance was found statistically to increase with decreasing distance between peristaltic waves and the pylorus. We conclude that transpyloric flow tends to be blocked when antral contraction waves are within a "zone of influence" proximal to the pylorus, suggesting physiological coordination between pyloric and antral contractile activity. We further conclude that gastric emptying of nutrient liquids is primarily through the "pressure pump" mechanism controlled by pyloric opening during periods of relative quiescence in antral contractile wave activity.

Adult↗
PubMed2000

Store-operated calcium entry and increased endothelial cell permeability.

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.

Animals↗
PubMed2000

Targeted somatic mutagenesis in mouse epidermis.

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.

Animals↗
PubMed2000

Inhibition of ubiquitin-proteasome pathway-mediated I kappa B alpha degradation by a naturally occurring antibacterial peptide.

Induction of NF-kappaB-dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the alpha 7 subunit of the 26S proteasome and blocks degradation of NF-kappa B inhibitor I kappa B alpha by the ubiquitin-proteasome pathway without affecting overall proteasome activity. I kappa B alpha phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of I kappa B alpha degradation abolishes induction of NF-kappa B-dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-kappa B-dependent gene expression for therapeutic purposes.

Animals↗
PubMed2000

Vagus nerve stimulation treatment for Lennox-Gastaut syndrome.

Lennox-Gastaut syndrome is a severe age-specific epilepsy syndrome that presents with medication-resistant seizures in childhood. Antiepileptic drugs are the mainstay of treatment. Nonpharmacologic treatments include corpus callosum section and the ketogenic diet. However, no single treatment is safe and effective. We treated 13 patients with Lennox-Gastaut syndrome between the ages of 4 and 44 years (mean, 16.7 years) with vagus nerve stimulation. During the first 6 months of treatment, vagus nerve stimulation produced a median seizure rate reduction of 52% (range, 0% to 93%; P = .04). At 6 months of follow-up, three patients had a greater than 90% reduction in seizures, two had a greater than 75% reduction, one had a greater than 50% reduction, and six had at least a 25% reduction. One patient did not improve. No patient worsened after initial improvement. Side effects, including hoarseness, coughing, and pain in the throat, were transient and tolerable. No patient discontinued vagus nerve stimulation. Our results suggest that vagus nerve stimulation could be an effective and safe adjunct therapy for the treatment of Lennox-Gastaut syndrome.

Adolescent↗