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Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 505 records · Page 28Linked to original sources
PubMed2000

Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples.

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.

Chromatography, High Pressure Liquid↗
PubMed2000

Relating helix tilt in a bilayer to lipid disorder: a mean-field theory.

We present a mean-field theory relating the helix tilt angle in a bilayer to lipid disorder. The theory provides a method to compare the rotational barriers for different helices in lipid bilayers. The results suggest that the helix tilt angle is strongly affected by both the hydrophobicity of the helix and the average lipid disorder. This leads us to point out future experiments that could shed light on lipid-protein interactions.

Algorithms↗
PubMed2000

Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells.

Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1beta receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK.

Adaptation, Physiological↗
PubMed2000

Protein concentration and enzyme digestion on microbeads for MALDI-TOF peptides mass mapping of proteins from dilute solutions.

A method for generating peptide mass maps from dilute protein samples is presented. The method involves the concentration of proteins from aqueous solution by adsorption onto reversed-phase polymeric microbeads. These beads are then washed extensively to remove contaminants, after which the bound proteins are digested with trypsin. Analysis of the digestion products is performed by MALDI-TOF mass spectrometry following direct deposition of the beads on a MALDI target, along with the matrix solution. The procedure is demonstrated using solutions of cytochrome c, lysozyme, and bovine serum albumin. The results of these digests are compared to trypsin digestions of the protein samples without sample preconcentration. Comparative results are also presented for protein solutions contaminated with 2 M NaCl, 2 M urea, or sodium dodecyl sulfate at concentrations up to 0.02%. These results reveal that, with the microbead preconcentration procedure, peptide mass maps can routinely be generated from highly contaminated samples with a protein concentration of only 100 nM.

Hydrolysis↗
PubMed2000

Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.

We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.

Adult↗
PubMed2000

Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons.

The Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase export Ca(2+) from the cytosol to the extracellular space. Three NCX genes (NCX1, NCX2, and NCX3), encoding proteins with very similar properties, are expressed at different levels in tissues. Essentially, no information is available on the mechanisms that regulate their expression. Specific antibodies have been prepared and used to explore the expression of NCX1 and NCX2 in rat cerebellum. The expression of NCX2 became strongly up-regulated during development, whereas comparatively minor effects were seen for NCX1. This was also observed in cultured granule cells induced to mature in physiological concentrations of potassium. By contrast, higher K(+) concentrations, which induce partial depolarization of the plasma membrane and promote the influx of Ca(2+), caused the complete disappearance of NCX2. Reverse transcription-polymerase chain reaction analysis showed that the process occurred at the transcriptional level and depended on the activation of the Ca(2+) calmodulin-dependent protein phosphatase, calcineurin. The NCX1 and NCX3 genes were also affected by the depolarizing treatment: the transcription of the latter became up-regulated, and the pattern of expression of the splice variants of the former changed. The effects on the NCX1 and NCX3 genes were calcineurin-independent.

Animals↗
PubMed2000

Caveolin-1 is regulated by c-myc and suppresses c-myc-induced apoptosis.

Recent data indicating that overexpression of caveolin-1 as well as c-myc are relatively common features of advanced prostate cancer prompted us to test for potential cooperative interactions between caveolin-1 and c-myc that would be consistent with malignant progression. We used the well-characterized Rat1AmycERT cells to show that the caveolin-1 gene is down-regulated at the level of transcription by c-myc. By maintaining relatively high levels of caveolin-1 with an adenoviral vector or in stably transfected clones we show that caveolin-1 can suppress c-myc-induced apoptosis. Further we established human prostate cancer cell lines with the mycER construct and show that clones with increased caveolin-1 are more resistant to myc-induced apoptosis and have increased capacity for growth in soft agar when c-myc is activated.

Animals↗
PubMed2000

Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential.

The involvement of 12-lipoxygenase (12-LOX) expression and function in tumor metastasis has been demonstrated in several murine tumor cell lines. In addition, 12-LOX expression was detected in human prostatic tumors and correlated to the clinical stage of disease. Here we provide data that human prostate cancer cell lines express the platelet-type isoform of 12-LOX at both the mRNA and protein levels, and immunohistochemistry revealed 12-LOX expression in human prostate tumors. The enzyme was localized to the plasma membrane, cytoplasmic organelles and nucleus in non-metastatic cells (PC-3 nm) and to the cytoskeleton and nucleus in metastatic cells (DU-145). After orthotopic/intraprostatic injection of tumor cells into SCID mice, the metastatic prostate carcinoma cells (DU-145) expressed 12-LOX at a significantly higher level compared with the non-metastatic counterparts, PC-3nm. The functional involvement of 12-LOX in the metastatic process was demonstrated when DU-145 cells were pretreated in vitro with the 12-LOX inhibitors N-benzyl-N-hydroxy-5-phenylpentamide (BHPP) or baicalein, the use of which significantly inhibited lung colonization. These data suggest a potential involvement of 12-LOX in the progression of human prostate cancer.

Animals↗
PubMed2000

Role of BK(Ca) channels and cyclic nucleotides in synergistic relaxation of trachea.

beta-Adrenoceptor agonists, nitric oxide (NO), and NO donors have been shown to mediate their effects through large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. The mechanism of the synergistic effect of the beta(2)-adrenoceptor agonist, salbutamol, and an NO donor, sodium nitroprusside, was studied in guinea pig tracheal preparations. Salbutamol (0.1 nM) and sodium nitroprusside (0.33 microM) alone relaxed the acetyl-beta-methylcholine chloride (methacholine)-contracted preparations only by 0.5% and 28%, respectively, but their combination caused a maximum of 60% relaxation (at 3 min), which stabilized to 40% (at 10 min). Iberiotoxin, a selective inhibitor of the BK(Ca) channels, did not abolish the synergistic effect. 3-isobutyl-1-methylxanthine (IBMX) did not modify relaxation evoked by the drugs. Concentrations of cyclic nucleotides did not correlate with relaxations as a function of time. The mechanism of synergy remains to be clarified. The results show that NO is an important modulator in the relaxation of guinea pig trachea induced by beta(2)-adrenoceptor agonists in vitro.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Structure-activity relationships of (E)-5-(2-bromovinyl)uracil and related pyrimidine nucleosides as antiviral agents for herpes viruses.

A series of (E)-5-(2-bromovinyl)uracil analogues and related nucleosides was synthesized, and their antiviral activities were evaluated. (E)-5-(2-Bromovinyl)-2'-deoxy-L-uridine (L-BVDU, 2), 1-(beta-L-arabinofuranosyl)-(E)-5-(2-bromovinyl)uracil (L-BVAU, 4), (E)-5-(2-bromovinyl)-1-(2-deoxy-2-fluoro-beta-L-ribofuranosyl)uracil (L-FBVRU, 8) and (E)-5-(2-bromovinyl)-1-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl)urac il (L-FBVAU, 10) were synthesized via appropriate 5-iodouracil analogues from L-arabinose. D- and L-Oxathiolane and -dioxolane derivatives 13, 16, 20, 21, and 29-34 were prepared by glycosylation reaction of the oxathiolane and dioxolane intermediates with silylated uracil analogues using TMSI as the coupling agent. The synthesized compounds were evaluated in cell cultures infected with the following viruses: varicella zoster virus (VZV), Epstein Barr virus (EBV), and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). Among the tested compounds, beta-L-CV-OddU (29), beta-L-BV-OddU (31), and beta-L-IV-OddU (33) exhibited potent in vitro antiviral activity against VZV with EC(50) values of 0.15, 0. 07, and 0.035 microM, respectively, and against EBV with EC(50) values of 0.49, 0.59, and 3.91 microM, respectively.

Antiviral Agents↗
PubMed2000

Positional genomic analysis identifies the beta(2)-adrenergic receptor gene as a susceptibility locus for human hypertension.

BACKGROUND: -After genome-wide linkage analyses of blood pressure levels, we resequenced 5 positional candidate genes in a linkage region on chromosome 5 and genotyped selected variants in several family samples from Rochester, Minn. METHODS AND RESULTS: In a sample of 55 pedigrees containing >/=1 sibling-pair(s) discordant for systolic blood pressure, polymorphisms within the beta(2)-adrenergic receptor gene (Arg16Gly, P=0.009) and the glutathione peroxidase 3 gene (-302G-->A, P=0.037; -623A-->C, P=0.013) were significantly related to blood pressure levels. In a second sample of 298 nuclear families (n=1283 individuals), the Arg16Gly polymorphism was significantly associated with diastolic blood pressure in family-based analyses (P=0.016) and with both diastolic (P=0.009) and mean arterial blood pressure (P=0.038) in analyses of the parental generation only. Neither polymorphism in the glutathione peroxidase 3 gene was associated with blood pressure levels in this sample. An additional 291 families (n=1240 individuals) were added to the nuclear family sample, and the Gln27Glu polymorphism in the beta(2)-adrenergic receptor gene was significantly associated with both systolic (P=0.034) and mean arterial blood pressure (P=0.035) in the parental generation of the combined 589 families. The frequencies of both the Gly16 and Glu27 alleles were higher in hypertensives than in normotensives (0.649 versus 0.604 and 0.490 versus 0.429, respectively), and the odds ratio for the occurrence of hypertension was 1.80 (95% confidence interval, 1.08 to 3.00; P=0. 023) for the Glu27 allele. CONCLUSIONS: The results of this study provide support for further detailed investigations of the mechanistic pathways by which variations in the beta(2)-adrenergic receptor gene may influence blood pressure levels.

Adolescent↗
PubMed2000

Transcriptional regulation of gene expression of sec6, a component of mammalian exocyst complex at the synapse.

Sec6, an essential component of the mammalian brain exocyst complex, is believed to function in synapse formation and synaptic plasticity. During neuronal development, the expression of the Sec6 gene correlates temporally with neurite outgrowth and synaptogenesis. To understand the mechanisms that regulate the Sec6 gene expression, we have cloned and characterized the 5'-terminal region of the murine Sec6 gene. We have shown that the 5'-untranslated region of the murine Sec6 gene is encoded by two exons that are separated by a 1560-bp intron. Primer extension analysis demonstrates that Sec6 gene transcription is initiated from a unique site. The Sec6 promoter is embedded in a CpG island and lacks canonical TATA or CAAT boxes. Sequence analysis of the 5'-flanking region and the first intron reveals the presence of a number of binding sites for transcription factors AP-1, AP-2, AP-4, ATF, C/EBPbeta, GATA-1, Oct 1, SP1, STAT, and NRSF. Transfection experiments using Sec6-luciferase fusion genes demonstrate that the 5'-flanking sequence functions as a strong promoter in neuronal but not in nonneuronal cells. Deletion analysis reveals the presence of a core promoter between nucleotide position -139 and +53, and two enhancer and four silencer elements within the 5'-flanking region and the first intron sequence. These results indicate that neuronal expression of the Sec6 gene involves a relatively specific core promoter and interplay between multiple positive and negative regulatory elements.

5' Untranslated Regions↗
PubMed2000

Coupling of peripheral tolerance to endogenous interleukin 10 promotes effective modulation of myelin-activated T cells and ameliorates experimental allergic encephalomyelitis.

Several immune-based approaches are being considered for modulation of inflammatory T cells and amelioration of autoimmune diseases. The most recent strategies include simulation of peripheral self-tolerance by injection of adjuvant free antigen, local delivery of cytokines by genetically altered T cells, and interference with the function of costimulatory molecules. Although promising results have been obtained from these studies that define mechanisms of T cell modulation, efficacy, practicality, and toxicity, concerns remain unsolved, thereby justifying further investigations to define alternatives for effective downregulation of aggressive T cells. In prior studies, we demonstrated that an immunoglobulin (Ig) chimera carrying the encephalitogenic proteolipid protein (PLP)1 peptide corresponding to amino acid sequence 139-151 of PLP, Ig-PLP1, is presented to T cells approximately 100-fold better than free PLP1. Here, we demonstrate that aggregation endows Ig-PLP1 with an additional feature, namely, induction of interleukin (IL)-10 production by macrophages and dendritic cells, both of which are antigen-presenting cells (APCs). These functions synergize in vivo and drive effective modulation of autoimmunity. Indeed, it is shown that animals with ongoing active experimental allergic encephalomyelitis dramatically reduce the severity of their paralysis when treated with adjuvant free aggregated Ig-PLP1. Moreover, IL-10 displays bystander antagonism on unrelated autoreactive T cells, allowing for reversal of disease involving multiple epitopes. Therefore, aggregated Ig-PLP1 likely brings together a peripheral T cell tolerance mechanism emanating from peptide presentation by APCs expressing suboptimal costimulatory molecules and IL-10 bystander suppression to drive a dual-modal T cell modulation system effective for reversal of autoimmunity involving several epitopes and diverse T cell specificities.

Animals↗
PubMed2000

Cloning and characterization of the murine pkd2 promoter.

Pkd2, the mouse homologue of PKD2, the gene responsible for the second form of autosomal dominant polycystic kidney disease, is highly expressed in fetal and adult mouse tissues. The expression of Pkd2 is developmentally regulated. To begin to dissect out the regulatory mechanism of Pkd2 expression, we characterized the basic features of the gene structure and identified potential cis-regulatory elements of Pkd2 transcription. Pkd2 spans 42 kb with a transcription start site 165 bp upstream of the translation start codon. Exon 1 of Pkd2 is 755 bp long, and the full-length transcript is 5215 bp. The Pkd2 promoter region is GC-rich and lacks a consensus TATA or CCAAT box. Consensus binding sites for the transcription factors Sp-1, NF-1, and Ap-2 lie in the 5' upstream region of Pkd2. The Sp-1 binding site is conserved in 5' upstream sequences of both the mouse and the human genes. The CAT activity of a series of upstream segments from +178 to -2749 was assessed in MDCK, LLCPK1, COS-7, and HEK293 cells. Deletion analysis identified a 409-bp fragment from position -221 to +178 responsible for basal promoter activity. A 922-bp fragment from -744 to +178 showed the highest level of CAT activity in the cell lines tested. These data define a functional promoter candidate region for Pkd2.

Animals↗
PubMed2000

Extensive sprouting of sensory afferents and hyperalgesia induced by conditional expression of nerve growth factor in the adult spinal cord.

Genetic transfer of growth-promoting molecules was proposed as a potential strategy to modify the nonpermissive nature of the adult CNS to induce axonal regeneration. To evaluate whether overexpression of neurotrophins or cellular adhesion molecules would effect axonal plasticity, adenoviruses encoding fibroblast growth factor-2 (FGF-2/Adts), nerve growth factor (NGF/Adts), neurotrophin-3, and the cell adhesion molecules N-cadherin and L1 were injected into the dorsal horn of the adult spinal cord. Transgene expression was primarily localized to astrocytes in the dorsal horn and motor neurons within the ventral horn. Overexpression of these factors, with the exception of NGF/Adts, failed to increase axonal sprouting. Eight days after NGF/Adts injections, axonal sprouting within the dorsal horn was apparent, and after 4 weeks, extensive spouting was observed throughout the entire dorsal horn, extending into the ventral horn and the white matter of the lateral funiculus. These axons were identified primarily as a subpopulation of nociceptive fibers expressing calcitonin gene-related peptide and substance-P. Behavioral analysis revealed thermal hyperalgesia and perturbation of accurate paw placement on grid-walking tasks for both FGF-2- and NGF-treated animals. These results indicate that the administration of growth-promoting molecules can induce robust axonal plasticity of normal adult primary sensory neurons into areas of transgene expression, causing significant alterations in behavioral responses. This observation also indicates that gene transfer protocols that aim to reconstruct diseased or injured pathways should also be designed to prevent the sprouting of the normal circuitry from adjacent unaffected neurons.

3T3 Cells↗
PubMed2000

Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain.

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn(2+)-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn(2+) bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.

Botulinum Toxins, Type A↗
PubMed2000

Molecular cloning of a novel water channel from rice: its products expression in Xenopus oocytes and involvement in chilling tolerance.

Water channel proteins, aquaporins, play a fundamental role in transmembrane water movements in plants. We isolated rice cDNA, rwc1, by screening a rice (Oryza sativa cv. Josaeng Tongil) cDNA library using a conserved motif of aquaporins. Like other aquaporin genes, rwc1 encodes a 290-residue protein with six putative transmembrane domains. The derived amino acid sequence of RWC1 shows high homology with PIP1 (plasma membrane intrinsic protein 1) subfamily members, which suggest it is localized in the plasma membrane. Injection of its cRNA into Xenopus oocytes increased the osmotic water permeability of the oocytes 2-3 times. Northern analysis showed that rice aquaporin genes are expressed in rice seedling leaves and roots, but that it disappeared from the root 6 h after osmotic stress began and that the transcript level remained low for about 24 h, then recovered. The time course of rice aquaporin gene-expression under osmotic stress was correlated with time course of turgor transition in plant. On the other hand, the levels of rice aquaporin gene-transcripts in leaves under chilling and recovery temperature depend on the pretreatment of mannitol for short time. This variation of the transcripts shown that rice aquaporin genes may play an important role in response to water stress-induced chilling tolerance.

Journal Article↗