Biomedical subjects

Jiali Li

Publications and source records attributed to Jiali Li.

18 recordsLinked to original sources

PubMed2006

Adenovirus fiber shaft contains a trimerization element that supports peptide fusion for targeted gene delivery.

Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

Adenoviridae↗

PubMed2005

Up-regulation of the JAK/STAT1 signal pathway during Chlamydia trachomatis infection.

Chlamydia trachomatis infection is the most common cause of sexually transmitted disease, leading to female pelvic inflammatory disease and infertility. The disease process has been linked to cellular response to this bacterial pathogen. This obligate intracellular pathogen infects macrophages, fibroblast cells, and epithelial and endothelial cells. We show in this study that infection of cervical epithelial cells, the primary target of Chlamydia trachomatis, leads to up-regulation and activation of the JAK/STAT signal pathway. Specifically, Chlamydia trachomatis infection of HeLa 229 cells selectively induces STAT1, STAT2, and IFN-stimulated transcription factor 3gamma expression and promotes STAT1 activation. The up-regulation of STAT1 is dependent on bacterial replication, because treatment of infected cells with antibiotics prevents STAT1 up-regulation. By analysis of the gene transcriptional and cytokine expression profiles of host cells combined with the use of neutralizing Abs, we show that IFN-beta production is critical for STAT1 induction in epithelial cells. Finally, we demonstrate that the host up-regulates STAT1 to restrict bacterial infection, because Chlamydia propagates more efficiently in STAT1-null or STAT1 knockdown cells, whereas Chlamydia growth is inhibited in cells with up-regulated STAT1 expression. This study demonstrates that the infected cells up-regulate the host innate antimicrobial response to chlamydial infection. It also highlights the importance of cellular response by nonimmune cells in host clearance of chlamydial infection.

Cell Line, Tumor↗

PubMed2005

Activation of tyrosine kinase of EGFR induces Gbetagamma-dependent GRK-EGFR complex formation.

This study demonstrated that activation of tyrosine kinase of epidermal growth factor receptor (EGFR) induces its association with G protein-coupled receptor kinase 2 (GRK2). Immunoprecipitation experiments showed that EGF stimulation increased GRK2 binding to EGFR complex in HEK293 cells coexpressing EGFR and GRK2. The EGF-induced GRK2-EGFR complex formation was greatly reduced by perturbation of EGFR and Src tyrosine kinase activity. Furthermore, studies with GRK2 mutants showed that neither catalytic activity nor the N-terminal domain of GRK2 was required for EGF-induced GRK2-EGFR complex formation. However, overexpression of Gbetagamma scavengers blocked EGF-induced formation of GRK2-EGFR complex.

Cell Line↗

PubMed2005

Slowing DNA translocation in a solid-state nanopore.

Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.

DNA↗

PubMed2005

Detecting single stranded DNA with a solid state nanopore.

Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.

DNA, Single-Stranded↗

PubMed2005

Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors.

Beta-arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signalling. Beta-arrestin1 and beta-arrestin2 preferentially bind to the phosphorylated GPCRs in response to agonist stimulation, resulting in receptor internalization and desensitization. The critical roles of GPCR kinases (GRKs)-catalyzed receptor phosphorylation and interaction of beta-arrestins with the phosphorylated receptor in receptor internalization are well established. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. The current study investigated the role of receptor phosphorylation and the involvement of different beta-arrestin subtypes in agonist-induced delta-opioid receptor (DOR) internalization in HEK293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labelling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and co-immunoprecipitation studies indicated that both the wild-type DOR and the phosphorylation-deficient mutant receptor could bind and recruit beta-arrestin1 and beta-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild-type and phosphorylation-deficient receptors was increased by overexpression of either type of beta-arrestins and blocked by dominant-negative mutants of beta-arrestin-mediated internalization, demonstrating that both phosphorylation-dependent and -independent internalization require beta-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either beta-arrestin1 or beta-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild-type DOR. However, agonist-induced internalization of the phosphorylation-deficient DOR was not affected by beta-arrestin1-specific RNAi but was blocked by RNAi against beta-arrestin2 subtype. These data indicate that endogenous beta-arrestin1 functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous beta-arrestin2, but not beta-arrestin1, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence of different requirement for beta-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent GPCR internalization.

Arrestins↗

PubMed2005

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinases (GRKs) mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors (GPCRs). We investigate the role of GRK2 on epidermal growth factor (EGF) receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase (ERK/MAPK) activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5-fold (P < 0.05) at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2, suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled delta opioid receptor internalization by approximately 40% (P < 0.01). Overall, these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.

Cell Line↗

PubMed2004

Regulation of complement C3 expression by the bile acid receptor FXR.

The farnesoid X receptor (FXR; NR1H4) is an intracellular bile acid-sensing transcription factor that plays a critical role in the regulation of synthesis and transport of bile acids as well as lipid metabolism. Although the reciprocal relationship between bile acid and triglyceride levels is well known, the mechanism underlying this link is not clearly defined. In this study, we demonstrate that FXR regulates the expression of at least two secreted factors, complement component C3 and FGF15, the rat ortholog of FGF19, known to influence lipid metabolism. The analysis of the human complement C3 gene reveals the presence of functional FXR response elements in the proximal promoter of C3. Furthermore, rats given a single dose of an FXR agonist exhibit an increase in the plasma concentration of complement C3 protein. These studies demonstrate a mechanism by which FXR, a nuclear receptor with a limited tissue expression pattern, regulates secretion of factors that ultimately can affect lipid metabolism in an endocrine or paracrine manner.

Animals↗

PubMed2004

chTNT-3/hu IL-12 fusion protein for the immunotherapy of experimental solid tumors.

Fusion proteins are emerging as a promising approach for targeting cytokines to the tumor site in order to generate an effective anti-tumor response. In this study, a fusion protein, chTNT-3/huIL-12, consisting of the necrosis targeting antibody, chTNT-3, and human interleukin-12 (IL-12), was constructed and expressed using the glutamine synthetase gene amplification system in NS0 cells. For these studies, IL-12 was chosen since it has been shown to be a powerful anti-tumor cytokine. To generate the fusion protein, an expression vector was prepared by linking the huIL-12 p35 subunit cDNA to the 3' end of the chTNT-3 heavy chain cDNA and the p40 subunit was added to a separate vector. The activity of the expressed chTNT-3/huIL-12 was confirmed by standard IL-12 bioactivity assays which demonstrated that the fusion protein induced similar levels of peripheral blood lymphocyte (PBL) proliferation as free recombinant IL-12. In addition, the lytic activity of the fusion protein was demonstrated in both naive and IL-2-activated lymphocytes using cytotoxicity assays against three human pancreatic and prostatic cancer cell lines (CAPAN, DU145, and PC3-MA). Human PBL incubated with this fusion protein showed an increase in IFN-gamma production which was augmented dramatically by pre-incubation with IL-2. Finally, the immunotherapeutic potential of the fusion protein was demonstrated in the human PBL-SCID mouse model where a 44% reduction in DU145 prostatic tumor growth was obtained compared to control treated mice. These results demonstrate that tumor-targeted human IL-12 may be an effective immunotherapeutic reagent for the treatment of solid tumors in man.

Animals↗

PubMed2003

Complete regression of experimental solid tumors by combination LEC/chTNT-3 immunotherapy and CD25(+) T-cell depletion.

LEC/chTNT-3, a chemokine fusion protein generated previously in our laboratory, produces a 40-60% reduction in well-established solid tumors of the BALB/c mouse. In this study, CD25(+) T-cell depletion was used in combination with LEC/chTNT-3 treatment to enhance the therapeutic value of this approach. In two tumor models (Colon 26 and RENCA), this combination immunotherapy produced complete regression of established s.c. tumors after 5 consecutive days of i.v. treatment. To show that targeted LEC is critical to these results, similar combination studies were performed with chTNT-3/cytokine fusion proteins consisting of human interleukin 2, murine IFN-gamma, and murine granulocyte macrophage colony-stimulating factor using identical treatment regimens. These studies showed no significant improvement indicating that combination therapy with anti-CD25(+) antisera requires LEC localization to tumor to produce complete regression. To study the mechanism of this remarkable response, immunotherapeutic studies were repeated in knockout mice and showed that successful treatment with CD25(+) depletion was dependent on the presence of IFN-gamma but not perforin. Other studies using real-time PCR, ex vivo proliferation, and intracellular cytokine staining with lymphocytes from tumor draining lymph nodes, suggested that this combination treatment was associated with increased T-helper 1 cytokine expression, enhanced T-cell activation, and increased IFN-gamma production by T cells. Rechallenge experiments showed that combination LEC/chTNT-3 treatment and CD25(+) depletion produced long-acting memory cells capable of preventing re-engraftment of the same but not different tumor cell lines. These studies suggest that LEC/monoclonal antibody fusion proteins, when used in combination with CD25(+) T-cell depletion, is a viable method of immunotherapy for the treatment of solid tumors.

Animals↗

PubMed2003

DNA molecules and configurations in a solid-state nanopore microscope.

A nanometre-scale pore in a solid-state membrane provides a new way of electronically probing the structure of single linear polymers, including those of biological interest in their native environments. Previous work with biological protein pores wide enough to let through and sense single-stranded DNA molecules demonstrates the power of using nanopores, but many future tasks and applications call for a robust solid-state pore whose nanometre-scale dimensions and properties may be selected, as one selects the lenses of a microscope. Here we demonstrate a solid-state nanopore microscope capable of observing individual molecules of double-stranded DNA and their folding behaviour. We discuss extensions of the nanopore microscope concept to alternative probing mechanisms and applications, including the study of molecular structure and sequencing.

DNA↗

PubMed2003

Identification of a protein fragment of interleukin 2 responsible for vasopermeability.

BACKGROUND: The cytokine interleukin 2 (IL-2) is involved in the activation of T cells and has been shown to play a central role in cancer immunotherapy. The full therapeutic potential of IL-2, however, has not been realized because of its dose-limiting systemic toxicity. We sought to identify a region of IL-2 that is responsible for the induction of vasopermeability (leaky tumor endothelium), a property associated with the toxicity of the molecule. METHODS: Intact IL-2 or overlapping synthetic peptides of IL-2 that were chemically conjugated to tumor-targeting monoclonal antibodies (TNT-1 or Lym-1) were injected into groups of mice (n = 4) that had previously been xenotransplanted with human tumor cells (ME-180 cervical carcinoma and Raji lymphoma). Two hours later, mice received intravenous injections of radiolabeled tracer antibody, and 3 days later they were subjected to biodistribution analysis to measure the ability of each immunoconjugate to enhance tumor uptake of the tracer antibody (i.e., vasopermeability activity). The cytokine activity of the immunoconjugates was determined by assaying their ability to promote the proliferation of a mouse IL-2-dependent cell line. RESULTS: Pretreatment of mice with an antibody/IL-2 immunoconjugate resulted in an approximately fourfold increase in radiolabeled tracer antibody uptake in the xenograft tumor as compared with uptake in mice injected with antibody alone. One synthetic fragment consisting of amino acids 22-58 contained 100% of the vasopermeability activity of IL-2 and was designated permeability-enhancing peptide (PEP). PEP had vasopermeability activity only when conjugated to a tumor-targeting antibody, had maximal activity as a dimer, and was devoid of cytokine activity. CONCLUSIONS: The identification of PEP should aid in the discovery of ways to decrease the toxicity of IL-2. Moreover, PEP is a promising candidate for the generation of agents that can enhance the delivery of antibodies and drugs to tumors.

Animals↗

PubMed2003

Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo.

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.

Animals↗

PubMed2003

Chimeric TNT-3 antibody/murine interferon-gamma fusion protein for the immunotherapy of solid malignancies.

Interferon-gamma (IFN-gamma) has been used in the experimental treatment of cancer with limited success. Despite direct cytotoxic effects on tumor cells and the ability to stimulate the antitumor activities of a variety of effector cells, IFN-gamma has not been found to produce impressive therapeutic responses partly because of inadequate sustained intratumoral concentrations and systemic toxicity. To overcome these obstacles, we have developed an antibody/murine IFN-gamma fusion protein (chTNT-3/muIFN-gamma), which utilizes the tumor necrosis therapy antibody, chTNT-3, to target murine IFN-gamma to necrotic regions of solid tumors implanted in immunocompetent BALB/c mice. The genetically engineered fusion protein was expressed in NS0 cells using the Glutamine Synthetase Gene Amplification Expression System. After purification, the fusion protein demonstrated both antigen targeting and cytokine activities as assessed by in vitro assays which, when compared to recombinant free IFN-gamma, demonstrated approximately 40-45% biologic activity by two separate assay determinations. Pharmacokinetic and biodistribution studies in mice demonstrated a relatively long whole body half-life of 32 h in vivo and significant intratumoral accretion, respectively. Most importantly, immunotherapeutic studies in the MAD109 syngeneic murine carcinoma of the lung demonstrated significant intratumoral infiltration by leukocytes, primarily by macrophages and CD4(-) CD8(-) Thy-1.2(+) lymphocytes. Additionally, intravenous administration of the fusion protein significantly decreased the number of metastatic foci in an experimental model of pulmonary metastasis without causing any observable toxicity. These studies demonstrate that chTNT3/muIFN-gamma can safely target syngeneic tumor models as part of a promising strategy for the targeted immunotherapy of solid tumors.

Animals↗

PubMed2002

Ion-beam sculpting time scales.

A study of ion sculpting dynamics in SiO2 and SiN using periodically pulsed ion beams reveals material transport that depends strongly on the time structure of the pulsed beams. It is found that significant nanoscale matter transport can occur over second long time scales after the ion beam has been extinguished. A simple phenomenological model described the dynamics of ion beam sculpting in terms of two material time scales. The model accounts for the surprising observation of enhanced matter transport affected by pulsed ion beams over continuous ion beam exposure.

Journal Article↗

PubMed2002

Regulation of cholesterol homeostasis and lipid metabolism in skeletal muscle by liver X receptors.

Recent studies have identified the liver X receptors (LXRalpha and LXRbeta) as important regulators of cholesterol and lipid metabolism. Although originally identified as liver-enriched transcription factors, LXRs are also expressed in skeletal muscle, a tissue that accounts for approximately 40% of human total body weight and is the major site of glucose utilization and fatty acid oxidation. Nevertheless, no studies have yet addressed the functional role of LXRs in muscle. In this work we utilize a combination of in vivo and in vitro analysis to demonstrate that LXRs can functionally regulate genes involved in cholesterol metabolism in skeletal muscle. Furthermore we show that treatment of muscle cells in vitro with synthetic agonists of LXR increases the efflux of intracellular cholesterol to extracellular acceptors such as high density lipoprotein, thus identifying this tissue as a potential important regulator of reverse cholesterol transport and high density lipoprotein levels. Additionally we demonstrate that LXRalpha and a subset of LXR target genes are induced during myogenesis, suggesting a role for LXR-dependent signaling in the differentiation process.

Animals↗

PubMed2002

Effect of CTP:phosphocholine cytidylyltransferase overexpression on the mouse lung surfactant system.

CTP:phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the biosynthesis by type II pneumocytes of phosphatidylcholine (PC), the predominant phospholipid in lung surfactant. Augmentation of endogenous CT activity might therefore result in enhanced surfactant PC production. To test this hypothesis, transgenic mice were created in which rat CT (rCT) was expressed under control of the human surfactant protein C (SP-C) promoter. Transgenic mice were identified by tail-clip PCR analysis and studies of four founder lines were initiated. Lung CT gene expression was enhanced in two transgenic founder lines relative to wild-type controls. These two transgenic lines also exhibited significantly higher levels of immunoreactive CT protein and CT activity in whole-lung homogenates and in cultured type II cell extracts. Disaturated PC (DSPC) content in whole-lung homogenates and the rate of DSPC synthesis in cultured type II cells were significantly increased in one transgenic line. However, neither the incorporation of radiolabeled precursors (choline and palmitate) into DSPC in vivo nor the cellular metabolism of DSPC differed significantly between transgenic and control mice. This transgenic model provides opportunity for further study of factors controlling surfactant phospholipid production in vivo.

Animals↗

PubMedDate not supplied

LEC/chTNT-3 fusion protein for the immunotherapy of experimental solid tumors.

The human chemokine liver-expression chemokine (LEC) was originally found in an expressed sequence tag library, and later the LEC gene was located to chromosome 17q in the ML chemokine gene cluster. LEC has been shown to chemoattract monocytes, lymphocytes, and polymorphonuclear leukocytes (PMNs) by its binding to CCR1 and CCR8 chemokine receptors. Because of its potency as a chemoattractant for immune cells, LEC was used to genetically engineer a fusion protein with chTNT-3, a monoclonal antibody previously shown to target tumors by binding to DNA exposed in necrotic zones. Because the N-terminus of chemokines is important for their activity, the C-terminus of LEC was genetically linked to the chTNT-3 heavy chain variable region and, along with the light chain gene, cotransfected into NSO murine myeloma cells using the glutamine synthetase gene amplification system. The expressed LEC/chTNT-3 fusion protein was purified by tandem protein-A affinity and ion-exchange chromatography and chemotaxis and binding assays confirmed the bioactivity of the purified fusion protein. Pharmacokinetic and biodistribution studies in vivo showed that LEC/chTNT-3 had a biologic half-life of 3 hours and had good uptake in tumor (2.4% injected dose/g), which remained stable at 12 and 24 hours postinjection. Immunotherapy studies performed in three solid tumor models of the BALB/c mouse showed between 37% and 55% tumor reduction at 19 days post-implantation. Immunohistochemical studies using tumor sections obtained at different time points after the administration of control chTNT-3 and LEC/chTNT-3 showed heavy infiltration of CD4+ and CD8+ T cells, PMNs, B cells, and CD11c+CD11b+ dendritic cells in the LEC/chTNT-3 treated groups. The results of these studies demonstrate that this novel fusion protein has potent antitumor activity that is associated with the infiltration of different subpopulations of immune cells. The targeting of LEC to necrotic areas of tumors where the release of tumor antigens is prevalent may be a new approach for the immunotherapy of solid tumors.

Animals↗