Biomedical subjects

Song Ling

Publications and source records attributed to Song Ling.

4 recordsLinked to original sources

PubMed2006

Sphingosine kinase 1-mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells.

OBJECTIVE: It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls. METHODS: Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1-phosphate (S1P), and by investigating the ability of the SPHK inhibitor N,N-dimethylsphingosine and isozyme-specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations. RESULTS: LCLs from patients with RA displayed disease-specific Fas-mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real-time PCR analysis showed higher SPHK-1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK-1 (but not SPHK-2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fas-induced apoptosis. Additionally, resistance to Fas-mediated signaling was reversed by siRNA oligonucleotides specific for SPHK-1 but not by oligonucleotides specific for SPHK-2. CONCLUSION: These findings demonstrate disease-specific resistance to Fas-mediated death signaling in patients with RA and implicate increased SPHK-1 activity as the cause of this aberration.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

OBJECTIVE: To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. METHODS: The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. RESULTS: In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. CONCLUSION: Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed2006

Activation of nitric oxide signaling by the rheumatoid arthritis shared epitope.

OBJECTIVE: Susceptibility to rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles encoding a shared epitope (SE) in positions 70-74 of the HLA-DRbeta chain. The mechanistic basis for this association is unknown. Given the proposed pathogenic role of nitric oxide (NO) in RA, this study was undertaken to examine whether the SE can trigger NO signaling events. METHODS: The intracellular levels of NO were measured with the fluorescent NO probe 4,5-diaminofluorescein diacetate and by the 2,3-diaminonaphthalene method. NO synthase activity was determined by measuring the rate of conversion of radioactive arginine to citrulline. Levels of cGMP were measured with a commercial enzyme-linked immunosorbent assay, and the cytolytic activity of T cells was measured using a standard (51)Cr release assay. RESULTS: Lymphoblastoid B cell lines carrying SE-positive HLA-DR alleles displayed a higher rate of spontaneous NO production compared with SE-negative cells. L cell transfectants expressing SE-positive DR molecules on their surface also generated higher levels of NO. Tetrameric HLA-DR molecules containing a DRbeta-chain encoded by the SE-positive DRB1*0401 allele stimulated fibroblast cells to produce higher levels of NO compared with cells stimulated with a control HLA-DR tetramer. Multimeric hepatitis B core proteins engineered to express region 65-79 encoded by the DRB1*0401 allele, but not the same region encoded by the control allele DRB1*0402, stimulated NO production in fibroblasts. Similarly, synthetic 15-mer peptides corresponding to the region 65-79 encoded by SE-positive alleles triggered increased NO levels when incubated with class II major histocompatibility complex-negative cells. The signaling pathway was found to involve NO synthase activation, followed by increased production of cGMP. SE-triggered increased NO levels inhibited cytolytic elimination of target cells. CONCLUSION: The SE can trigger NO-mediated signaling events in opposite cells, and may thereby contribute to RA pathogenesis.

Amino Acid Sequence↗

PubMed2004

Genoprotective pathways. II. Attenuation of oxidative DNA damage by isopentenyl diphosphate.

Oxidative stress is believed to play a role in the pathogenesis of many diseases. Here we report that isopentenyl diphosphate (IPP), the 5-carbon building unit of all isoprenoids, is a potent antioxidant that is capable of inhibiting oxidative DNA damage at picomolar concentrations (IC50 = 1.7 x 10(-11) M). The diphosphate moiety is essential, since isopentenyl monophosphate (IMP) is unable to trigger antioxidative signaling. The 20-carbon isoprenyl, geranylgeranyl diphosphate (GGPP), but not the 15-carbon farnesyl diphosphate, displays similar genoprotective effects. The pathway activated by IPP is distinct from that of 2-chloroadenosine (2CA). 2CA-mediated genoprotective signaling is transduced through an A2a or A2b adenosine receptor (AR) and can be blocked by the cyclic AMP (cAMP)-dependent protein kinase (PKA) inhibitor, H-89. In contrast, IPP signaling is independent of A2aAR, A2bAR, cAMP or PKA. Unlike the 2CA-mediated pathway, the effect of IPP is dependent on the mevalonate pathway, a geranylgeranylated protein and on intact proteasome activity. Thus, IPP is a potent activator of a novel genoprotective pathway. These findings shed new light on the role of isoprenoids in oxidative stress biology and may help to develop novel preventive strategies against oxidative damage.

Antimutagenic Agents↗