Biomedical subjects

Fengling Li

Publications and source records attributed to Fengling Li.

9 recordsLinked to original sources

PubMed2025

Development of a chemical probe to enable characterization of the casein kinase 1γ subfamily.

The casein kinase 1γ (CK1γ) subfamily, while severely understudied, is implicated in diverse disease-relevant pathways, including WNT signaling and human cytomegalovirus (HCMV) replication. While genetic tools exist to study CK1γ, the selective inhibition of CK1γ through pharmacological means remains underexplored. Chemical probes, or potent and selective inhibitors, remain one of the most powerful pharmacological tools for uncovering protein biology. Herein, we developed several novel assays for assessing target engagement with the CK1γ subfamily in cells. Enabled by these assays, we conducted a comprehensive structure-activity relationship (SAR) campaign to develop the first chemical probe, SGC-CK1γ-1, for the CK1γ subfamily. SGC-CK1γ-1, which was developed alongside a structurally related negative control compound, potently and selectively inhibited the CK1γ kinases in living cells, plus inhibited both WNT signaling and human cytomegalovirus replication.

Journal Article↗

PubMed2007

An efficient method for producing an indexed, insertional-mutant library in rice.

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.

Chromosome Mapping↗

PubMed2005

Transcriptional profiling of imbibed Brassica napus seed.

Using an Arabidopsis microarray, we compared gene expression between germinating Brassica napus seeds and seeds in which germination was inhibited either by polyethylene glycol (PEG) or by the abscisic acid (ABA) analog PBI429, which produces stronger and longer lasting ABA-like effects. A total of 40 genes were induced relative to the germinating control by both treatments. Conspicuous among these were genes associated with late seed development. We identified 36 genes that were downregulated by both PEG and PBI429. Functions of these genes included carbohydrate metabolism, cell wall-related processes, detoxification of reactive oxygen, and triacylglycerol breakdown. The PBI429 treatment produced an increase in endogenous ABA and increased ABA catabolism. However, PEG treatment did not result in similar effects. The transcription factor ABI5 was consistently upregulated by both treatments and PKL was downregulated. These results suggest a greater importance of ABA signaling and reduced importance of GA signaling in nongerminating seeds.

Abscisic Acid↗

PubMed2003

Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells.

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-kappaB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-kappaB DNA binding to the p40 promoter and inhibits LPS-induced IkappaB phosphorylation. Similarly, NO attenuates IL-1beta-induced NF-kappaB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.

Animals↗

PubMed2003

Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP.

Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-gamma and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at -68 to -54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-gamma. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-gamma-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-gamma-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.

Animals↗

PubMed2003

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation.

Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.

Animals↗

PubMed2002

Chronic intestinal inflammation and angiogenesis in genetically susceptible rats is modulated by kininogen deficiency.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis associated with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK), we backcrossed Brown Norway rats having an HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome. The HK values of F5WT rat plasma and F5HKd rat plasma were 0.62 +/- 0.20 and 0.08 +/- 0.03 U/ml, respectively. Among the inflammatory changes, the mean gross gut, total intestinal histologic and liver granuloma score and the white blood count were significantly lower in the F5HKd than the F5WT rats. Plasma T-kininogen was significantly less in F5HKd. Angiogenesis (mean vascular density) in the cecum was decreased significantly in F5HKd compared to F5WT. These results indicate the importance of the kallikrein-kinin system in this model of chronic enterocolitis and systemic inflammation.

Animals↗

PubMed2002

Inflammatory-mediated repression of the rat ileal sodium-dependent bile acid transporter by c-fos nuclear translocation.

BACKGROUND & AIMS: Ileal malabsorption of bile salts is observed in Crohn's ileitis. We define the transcriptional mechanisms involved in cytokine-mediated repression of the rat apical sodium-dependent bile acid transporter (ASBT). METHODS: ASBT regulation was studied in IL-1beta-treated IEC-6 and Caco-2 cells and in indomethacin-treated rats. RESULTS: Indomethacin-induced ileitis in Lewis rats leads to specific reductions in ileal ASBT messenger RNA and protein levels, whereas c-jun and c-fos are induced. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor repress the activity of the ASBT promoter in Caco-2 and intestinal epithelial cell-6 cells. This effect is blocked by the proteasome inhibitor, MG-132, or by the phosphatidyl inositol 3-kinase inhibitor, wortmannin. Indomethacin (in vivo) or proinflammatory cytokine (in vitro) treatment leads to serine phosphorylation and nuclear translocation of c-fos. Mutation of a 5' activated protein (AP)-1 site inactivates the ASBT promoter, whereas mutation of the 3' site abrogates the proinflammatory cytokine-mediated repression. The 5' site binds a c-jun homodimer, whereas the 3' site binds a c-jun/c-fos heterodimer. c-Jun overexpression enhances ASBT promoter activity, whereas a dominant negative c-jun construct inactivates the promoter. c-Fos overexpression represses promoter activity. A 27 base pair cis-element from the 3' site in the ASBT promoter imparts cytokine-mediated down-regulation to a heterologous SV40 promoter construct. CONCLUSIONS: The ASBT promoter contains 2 distinct cis AP-1 elements; the 5' element binds homodimeric c-jun and mediates basal transcription. Inflammation is associated with up-regulation, phosphorylation, and nuclear translocation of c-fos, which then represses ASBT promoter activity via binding of the 3' AP-1 element by a c-fos/c-jun heterodimer.

Androstadienes↗

PubMed2002

Kininogen deficiency modulates chronic intestinal inflammation in genetically susceptible rats.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis concomitant with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK) in chronic enterocolitis, we back crossed Brown-Norway rats having a HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome. The HK values of F5WT and F5HKd rat plasma were 0.62 +/- 0.20 and 0.08 +/- 0.03 U/ml, respectively. In PG-APS-injected rats, chronic inflammation was measured by using gross gut score, histological inflammation, liver granuloma, and white blood cell count. The mean gross gut scores were significantly lower in the F5HKd than in the F5WT rats. Plasma T-kininogen was significantly less in F5HKd. These results indicate the importance of the kallikrein-kinin system in this model of chronic enterocolitis and systemic inflammation.

Animals↗