Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.
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PMID:Fluobodies: green fluorescent single-chain Fv fusion proteins. 1059 59

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.
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PMID:Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. 1074 19

The effect of mite antigens on murine lymphocytes and macrophages was studied in vitro. Antigens prepared from Dermatophagoides farinae bodies (Dfb) or recombinant Mag3, glutathione-S transferase (GST)-fused mite antigen, stimulated murine spleen cells to proliferate. The responder cells were B cells, because the response was sensitive to anti-Ig antibody and C treatment, but not to anti-Thy 1 antibody and C treatment. The response was not due to lipopolysaccharide contamination, a representative B-cell mitogen, because polymyxin B column-passed Dfb significantly stimulated B cells, and GST protein alone did not stimulate them. Alloantigen presenting activity was increased in mite antigen-treated B cells and spleen adherent cells. Mite antigens stimulated CD80 and the major histocompatibility complex (MHC) class II molecule expression, but suppressed CD86 expression on B cells and spleen adherent cells that were detected by a flow cytofluorometer. Antibodies to the MHC class II molecules, CD80 and CD86 blocked the alloantigen-presenting activity. Furthermore, mite antigens stimulated B cells and spleen adherent cells to produce cytokines. These results suggest that mite antigens have a stimulating activity on antigen-presenting cells/macrophages and modulate immune responses.
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PMID:Effect of mite antigens on antigen presenting cells/macrophages in mice. 1101 87

The effect of cytokines, lipopolysaccharide, and ethanol on inducible nitric-oxide synthase (iNOS) expression was studied in C6 glial cells. Maximal induced activity, measured by the accumulation of nitrite in culture medium, occurred following treatment with lipopolysaccharide and interferon-gamma. Each cytokine alone was ineffective, whereas an optimal combination of interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma was near maximal, indicating synergistic interactions. Other combinations caused submaximal activity. Ethanol is known to suppress iNOS expression in C6 cells induced by a phorbol ester plus lipopolysaccharide. The current work shows ethanol also suppresses cytokine-induced iNOS expression and reduces interleukin-1beta and tumor necrosis factor-alpha potency without affecting interferon-gamma potency. Ethanol-mediated reductions in cytokine-induced iNOS mRNA and immunoreactive protein levels suggested an effect on gene transcription. Therefore, C6 cells stably expressing 1846 and 526 base fragments of the rat iNOS gene promoter fused to a luciferase reporter gene were prepared and characterized and used to study the effect of ethanol on iNOS promoter activity. Promoter activity in stable transfected C6 cells was inhibited by ethanol exposure with a similar concentration dependence as observed for inhibition of nitrite production, indicating that iNOS inhibition by ethanol is transcriptional. Furthermore, ethanol inhibition of the 526 base fragment activity, which lacks interferon-gamma enhancement of lipopolysaccharide-induced luciferase activity, confirmed that interferon-gamma-responsive elements do not participate in acute ethanol-induced inhibition of rat iNOS gene transcription.
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PMID:Cytokine-induced iNOS expression in C6 glial cells: transcriptional inhibition by ethanol. 1145 39

Tissue-type plasminogen activator (t-PA) participates in the control of synaptic plasticity and memory formation in the central nervous system (CNS). Transgenic mice harbouring either 9.5, 3.0 or 1.4 kb of the human t-PA promoter fused to the LacZ reporter gene were used to assess t-PA promoter-directed expression in vivo. The 9.5 kb t-PA promoter directed expression to the brain, most notably to the dentate gyrus, superior colliculus, hippocampus, thalamus and piriform cortex. Staining was also observed in the retrosplenial and somatosensory cortex. The 3.0 kb t-PA promoter directed generalized and poorly defined expression to the cortex and hippocampus, while the 1.4 kb t-PA promoter directed expression selectively to the medial habenula. Intravenous administration of lipopolysaccharide into mice harbouring the 9.5 kb t-PA promoter resulted in an increase in reporter gene activity in the lateral orbital cortex and thalamus. Results of in vitro transfection experiments of NT2 cells with a series of t-PA promoter deletion constructs confirmed the presence of regulatory elements throughout the 9.5 kb promoter region. Finally, we describe a cis-acting element related to the NFAT recognition site that provides a protein-binding site and which may play a role in the selective expression of the 1.4 t-PA promoter in the medial habenula. These results indicate that elements between -3.0 and -9.5 kb of the t-PA promoter confer constitutive and inducible expression to specific regions of the CNS.
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PMID:Control elements between -9.5 and -3.0 kb in the human tissue-type plasminogen activator gene promoter direct spatial and inducible expression to the murine brain. 1157 84

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated them ES-DCs (ES cell-derived dendritic cells), and the functions of ES-DCs were comparable with those of DCs generated from bone marrow cells. Upon transfer to new dishes and stimulation with interleukin-4 plus tumor necrosis factor alpha, combined with anti-CD40 monoclonal antibody or lipopolysaccharide, ES-DCs completely became mature DCs, characterized by a typical morphology and higher capacity to stimulate MLR. Using an expression vector containing the internal ribosomal entry site-puromycin N-acetyltransferase gene or a Cre-lox-mediated exchangeable gene-trap system, we could efficiently generate ES cell transfectants expressing the products of introduced genes after their differentiation to DCs. ES-DCs expressing invariant chain fused to a pigeon cytochrome C epitope presented the epitope efficiently in the context of E(k). We primed ovalbumin (OVA)-specific cytotoxic T lymphocytes in vivo by injecting mice with ES-DCs expressing OVA, thus demonstrating immunization with ES-DCs genetically engineered to express antigenic protein. The methods may be applicable to immunomodulation therapy and gene-trap investigations of DCs.
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PMID:Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells. 1240 78

CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.
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PMID:Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen. 1254 58

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coil maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.
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PMID:The heat shock proteins, Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells. 1265 78

L-carnitine is an essential nutrient with a major role in cellular energy production. There is evidence that, at high doses, L-carnitine might mimic some of the biological activities of glucocorticoids, especially immunomodulation. To explore the molecular basis of this effect, we tested the influence of L-carnitine on glucocorticoid receptor-alpha (GRalpha) functions. Millimolar concentrations of L-carnitine, which were not cytotoxic in vitro, significantly reduced the whole cell binding of [3H]dexamethasone to GRalpha by decreasing the affinity of this receptor for its steroid ligand. At the same concentrations, L-carnitine was able to trigger nuclear translocation of green fluorescent protein (GFP)-fused human GRalpha and transactivate the glucocorticoid-responsive mouse mammary tumor virus (MMTV) and TAT3 promoters in a dose-dependent fashion. This effect was solely dependent on the presence of glucocorticoid-responsive elements on the promoter and on the expression of functional GRalpha by the cell. Finally, similarly to glucocorticoids, L-carnitine suppressed tumor necrosis factor-alpha (TNFalpha) and interleukin-12 release by human primary monocytes stimulated with lipopolysaccharide ex vivo. Both GRalpha transactivation and cytokine suppression by L-carnitine were abrogated by the GRalpha-antagonist RU 486. Taken together, our results suggest that pharmacological doses of L-carnitine can activate GRalpha and, through this mechanism, regulate glucocorticoid-responsive genes, potentially sharing some of the biological and therapeutic properties of glucocorticoids.
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PMID:L-carnitine: A nutritional modulator of glucocorticoid receptor functions. 1282 92

We have studied the anti-inflammatory activity of (6-(p-bromophenyl)amino-7-(p-chlorophenyl)indazolo[2',3':1,5]-1,2,4-triazolo[4,3-a]-1,3,5-benzotriazepine (ITB), prepared by solid-phase synthesis. This novel compound reduced the production of nitrite and PGE(2) in RAW 264.7 macrophages stimulated with lipopolysaccharide in a concentration-dependent manner. The first effect was dependent on inhibition of nitric oxide synthase-2 (NOS-2) protein expression although ITB did not modify nuclear factor-kappa B (NF-kappa B)-DNA binding. In addition, this compound inhibited cyclooxygenase-2 (COX-2) activity, which was also observed in aspirin-treated human monocytes. The anti-inflammatory effects of ITB were demonstrated in the carrageenan-induced mouse paw oedema, where this compound inhibited swelling and PGE(2) levels in inflamed paws but not in stomachs, in contrast to the dual COX-1/COX-2 inhibitor indomethacin. Inhibition of COX-2 activity and NOS-2 protein expression was confirmed in vivo using the zymosan-injected mouse air pouch model. These results suggest that synthesis of fused indazolo bis(guanidines) is a useful approach in the search for new anti-inflammatory agents.
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PMID:A novel indazolo-triazolo-benzotriazepine exerts anti-inflammatory effects by inhibition of cyclooxygenase-2 activity and nitric oxide synthase-2 expression. 1282 17


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