Induced Expression (induced + expression)

Distribution by Scientific Domains
Distribution within Medical Sciences


Selected Abstracts


DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought-Induced Male Sterility in Rice Anthers,

JOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2010
G. N. Nguyen
Abstract Rice plants exposed to three consecutive days of water stress (,0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT-PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers. [source]


Activation of caspase-3 alone is insufficient for apoptotic morphological changes in human neuroblastoma cells

JOURNAL OF NEUROCHEMISTRY, Issue 6 2002
Margaret M. Racke
Abstract Activated caspase-3 is considered an important enzyme in the cell death pathway. To study the specific role of caspase-3 activation in neuronal cells, we generated a stable tetracycline-regulated SK-N-MC neuroblastoma cell line, which expressed a highly efficient self-activating chimeric,caspase-3, consisting of the caspase-1 prodomain fused to the caspase-3 catalytic domain. Under expression-inducing conditions, we observed a time-dependent increase of processed caspase-3 by immunostaining for the active form of the enzyme, intracellular caspase-3 enzyme activity, as well as poly(ADP-ribose) polymerase (PARP) cleavage. Induced expression of the caspase fusion protein showed predominantly caspase-3 activity without any apoptotic morphological changes. In contrast, staurosporine treatment of the same cells resulted in activation of multiple caspases and profound apoptotic morphology. Our work provides evidence that auto-activation of caspase-3 can be efficiently achieved with a longer prodomain and that neuronal cell apoptosis may require another caspase or activation of multiple caspase enzymes. [source]


Axe,Txe, a broad-spectrum proteic toxin,antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium

MOLECULAR MICROBIOLOGY, Issue 5 2003
Ruth Grady
Summary Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin,antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM,yoeB) present in the E. coli chromosome function as a toxin,antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe,Txe is one of the first functional proteic toxin,antitoxin systems to be accurately described for Gram-positive bacteria. [source]


MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogen

PROTEOMICS - CLINICAL APPLICATIONS, Issue 5 2010
Monika Hermansson
Abstract Purpose: Citrullination is a post-translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past. Experimental design: Citrullination of human fibrinogen, a candidate autoantigen in arthritis, was studied. Accurate identification of citrullinated fibrinogen peptides from rheumatoid arthritis synovial tissue specimens was performed using accurate mass and retention time analysis. Results: A peptide with the sequence ESSSHHPGIAEFPSRGK corresponding to amino acids 559,575 of fibrinogen ,-chain was identified to be citrullinated with an occupancy rate between 1.4 and 2.5%. Citrullination of the peptide KREEAPSLRPAPPPISGGGYRARPAK corresponding to amino acids 52,77 of the fibrinogen ,-chain was identified with an occupancy rate of 1.2%. Conclusions and clinical relevance: We report a proof of principle study for the identification of citrullinated proteins and within them, identification of citrullination sites and quantification of their occupancies in synovial tissue from rheumatoid arthritis patients using high-resolution MS. Detailed studies on which molecules are citrullinated in arthritis can provide information about their role in immune regulation and serve as novel biomarkers and potentially even as therapeutic targets. [source]


Leishmania donovani -induced expression of signal regulatory protein , on Kupffer cells enhances hepatic invariant NKT-cell activation

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2010
Lynette Beattie
Abstract Signal regulatory protein , (SIRP,) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRP,,CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN-, from splenic iNKT cells following exposure to the ,GalCer analogue PBS-57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRP, was undetectable in the liver of uninfected mice, the hepatic iNKT-cell response to infection was also impaired in CD47,/, mice. However, we found that SIRP, was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G-protein-coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection. [source]


c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003
Orisa J. Igwe
Abstract Increased synthesis of substance P (SP) in the dorsal root ganglia (DRG) and enhanced axonal transport to and secretion from the primary afferent sensory neurons might enhance pain signalling in the spinal dorsal horn by modifying pronociceptive pathways. IL-1, increases SP synthesis by enhancing the expression of preprotachykinin (PPT) mRNA encoding for SP and other tachykinins in the DRG. Stimulation of IL-1 receptor by IL-1, may induce the phosphorylation of tyrosine residues in many effector proteins through the activation of p60c-src kinase. The hypothesis that the synthesis of SP in and secretion from the primary sensory ganglia are regulated by the activation of p60c-src kinase induced by IL-1, was tested. Pretreatment of DRG neurons in culture with herbimycin A, genistein or PP2, three structurally different nonreceptor tyrosine kinase inhibitors that act by different mechanisms, decreased the kinase activity of p60c-src induced by the activation of IL-1 receptor. PP3, a negative control for the Src family of tyrosine kinase inhibitor PP2 had no effect. Herbimycin A and genistein also decreased IL-1,-induced expression of PPT mRNA-encoding transcripts and the levels of SP-li synthesized in the cells and secreted into the culture medium in a concentration-dependent manner. SB 203580 [a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor] and PD 98059 (a p44/42 MAPK kinase inhibitor) were ineffective in modulating IL-1,-induced SP synthesis and secretion, and p60c-src kinase activity in DRG neurons. Whereas, IL-1 receptor antagonist and cycloheximide inhibited IL-1,-evoked secretion of SP-like immunoreactivity (SP-li), actinomycin D decreased it significantly but did not entirely abolish it. These findings show that phosphorylation of specific protein tyrosine residue(s) following IL-1 receptor activation might play a key role in IL-1, signalling to modulate PPT gene expression and SP secretion in sensory neurons. In view of the role of SP as an immunomodulator, these studies provide a new insight into neural-immune intercommunication in pain regulation in the sensory ganglia through the IL-1,-induced p60c-src activation. [source]


Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro

EXPERIMENTAL DERMATOLOGY, Issue 4 2000
J. K. Laihia
Abstract: It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12,24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8±0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon. [source]


Proteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sexta

FEBS JOURNAL, Issue 1 2010
Chunju An
The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal,ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and ,-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to ,-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract ,,MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807) [source]


Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa

FEBS JOURNAL, Issue 24 2001
Zhong Dong
C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection. [source]


Epidermal keratinocytes do not activate peripheral T-cells: interleukin-10 as a possible regulator

IMMUNOLOGY, Issue 3 2008
Rocío Isabel Domínguez-Castillo
Summary The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-, to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-,-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model. [source]


Sulphasalazine inhibits macrophage activation: inhibitory effects on inducible nitric oxide synthase expression, interleukin-12 production and major histocompatibility complex II expression

IMMUNOLOGY, Issue 4 2001
György Haskó
Summary The anti-inflammatory agent sulphasalazine is an important component of several treatment regimens in the therapy of ulcerative colitis, Crohn's disease and rheumatoid arthritis. Sulphasalazine has many immunomodulatory actions, including modulation of the function of a variety of cell types, such as lymphocytes, natural killer cells, epithelial cells and mast cells. However, the effect of this agent on macrophage (M,) function has not been characterized in detail. In the present study, we investigated the effect of sulphasalazine and two related compounds , sulphapyridine and 5-aminosalicylic acid , on M, activation induced by bacterial lipopolysaccharide (LPS) and interferon-, (IFN-,). In J774 M, stimulated with LPS (10 µg/ml) and IFN-, (100 U/ml), sulphasalazine (50,500 µm) suppressed nitric oxide (NO) production in a concentration-dependent manner. The expression of the inducible NO synthase (iNOS) was suppressed by sulphasalazine at 500 µm. Sulphasalazine inhibited the LPS/IFN-,-induced production of both interleukin-12 (IL-12) p40 and p70. The suppression of both NO and IL-12 production by sulphasalazine was superior to that by either sulphapyridine or 5-aminosalicylic acid. Although the combination of LPS and IFN-, induced a rapid expression of the active forms of p38 and p42/44 mitogen-activated protein kinases and c-Jun terminal kinase, sulphasalazine failed to interfere with the activation of any of these kinases. Finally, sulphasalazine suppressed the IFN-,-induced expression of major histocompatibility complex class II. These results demonstrate that the M, is an important target of the immunosuppressive effect of sulphasalazine. [source]


Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2004
Rosealee Smith
Abstract PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption. Introduction: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated. Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR. Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3 -induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells. Conclusion: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation. [source]


Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin-1, and ,

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010
D. Nowinski
Abstract Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-, and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF-,-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 ,-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1, and ,. Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1, or , in presence or absence of TGF-,1. IL-1 suppressed basal and TGF-,-induced CTGF mRNA and protein expression. IL-1, and , inhibited TGF-,-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements. Furthermore, IL-1, and , inhibited TGF-,-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF-, activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-,-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226,1233, 2010. Published 2010 Wiley-Liss, Inc. [source]


Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008
Christopher M. Amantea
Abstract Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of ,-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes. J. Cell. Biochem. 105: 424,436, 2008. © 2008 Wiley-Liss, Inc. [source]


Salvianolic acid B attenuates plasminogen activator inhibitor type 1 production in TNF-, treated human umbilical vein endothelial cells

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005
Zhe Zhou
Abstract Plasminogen activator inhibitor type 1 (PAI-1), which plays a role in the development of atherosclerosis, is produced by endothelial cells following stimulation with various inflammatory cytokines such as tumor necrosis factor (TNF-,). In the present study, we investigated the effects of a potent water-soluble antioxidant, salvianolic acid B (SalB; derived from the Chinese herb, Salviamiltiorrhiza), on the expression of PAI-1 in TNF-,-treated human umbilical vein endothelial cells (HUVECs). We found that SalB inhibited TNF-,-induced PAI-1 mRNA production and protein secretion in HUVECs. Treatment with SalB (0.05 and 0.15 µM) notably attenuated TNF-, induced expression of PAI-1 to 90.5% and 74.6%, respectively, after 12 h, and to 75.1% and 64.2%, respectively, after 18 h. We also observed a dose-dependent decrease in PAI-1 protein production in the presence of SalB. We then used pathway inhibitors to investigate which step of the TNF-, induced signaling pathway was targeted by SalB. We found that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, increased the inhibitory effects of SalB on TNF-,-induced PAI-1 secretion, whereas the nuclear factor-,B (NF-,B) inhibitor, emodin, and the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, did not. A gel shift assay further showed that SalB inhibited the TNF-,-activated NF-,B and AP-1 DNA binding activities in a dose-dependent manner. Collectively, these results indicate that the NF-,B and ERK-AP-1 pathways are possible targets of SalB in the regulation of TNF-,-stimulated PAI-1 production in HUVECs. © 2005 Wiley-Liss, Inc. [source]


Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-,-treated human aortic endothelial cells

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2001
Yung-Hsiang Chen
Abstract Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-, (TNF-,)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 ,g/ml), the TNF-,-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2,±,3.2% and 80.0,±,2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 ,g/ml), 84.5,±,1.9%, 78.8,±,1.2%, 58.9,±,0.4%, 58.7,±,0.9%, and 57.4,±,0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 ,g/ml) or Sal B (5 ,g/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-,-stimulated HAECs (45.7,±,2.5% and 55.8,±,1.2%, respectively). SME or Sal B significantly inhibited TNF-,-induced activation of nuclear factor kappa B (NF-,B) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis. J. Cell. Biochem. 82:512,521, 2001. © 2001 Wiley-Liss, Inc. [source]


Role of atypical protein kinase C isozymes and NF-,B in IL-1,-induced expression of cyclooxygenase-2 in human myometrial smooth muscle cells

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007
Sara V. Duggan
Increased myometrial expression of cyclooxygenase-2 (Cox-2) at term results from elevated local levels of inflammatory cytokines, and its inhibition provides a potential route for intervention in human pre-term labor. We have identified a role for atypical protein kinase C (PKC) isozymes in IL-1,-induced Cox-2 expression in human myometrial smooth muscle cells (HMSMC). The PKC inhibitor GF109203X (10 µM) inhibited IL-1,-induced Cox-2 protein and RNA expression, which were also reduced by MAPK and nuclear factor ,B (NF-,B) inhibitors. GF109203X did not affect MAPK activities, and neither did it replicate the effect of p38 MAPK inhibition on Cox-2 mRNA stability, suggesting that PKC operates through an independent mechanism. The effect of GF109203X remained intact after depletion of conventional and novel PKC isozymes by phorbol ester pre-treatment. In contrast LY379196 (10 µM), which at micromolar concentrations inhibits all but atypical PKCs, did not affect Cox-2 expression. A peptide corresponding to the pseudosubstrate sequence of atypical PKCs blocked Cox-2 protein expression, whereas the sequence from conventional PKCs was ineffective. GF109203X did not affect NF-,B binding to nuclear proteins, but strongly reduced NF-,B-dependent transcription in luciferase reporter assays. Our findings indicate that IL-1,-induced Cox-2 expression in HMSMC in culture requires p38-MAPK-mediated mRNA stabilization and an independent activation of Cox-2 transcription which is dependent on the action of atypical PKCs, probably through direct stimulation of the transactivating activity of NF-,B. J. Cell. Physiol. 210: 637,643, 2007. © 2006 Wiley-Liss, Inc. [source]


Non-invasive detection of the metabolic burden on recombinant microorganisms during fermentation processes,

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2001
Th Bachinger
Abstract Heterologous protein production is an important source of therapeutic products. Optimisation of such bioprocesses by adjustment of the expression rate of the heterologous protein to the biosynthetic capacity of the cell metabolism would benefit from an online method for monitoring the metabolic burden. In this study we evaluated the use of a chemical multi-sensor array for this purpose. Fermentations with a recombinant Escherichia coli strain expressing human superoxide dismutase (rhSOD) were monitored by the sensor array. The results of isopropyl-thiogalactopyranoside (IPTG)-induced expression were compared with fermentations with a plasmid-free strain. The overproduction of rhSOD, imposing a high metabolic burden on the plasmid-carrying cells, was distinctly and reproducibly observed by the multi-sensor array. The potential of this non-invasive method of non-specific metabolic burden monitoring is demonstrated by the results of the study. © 2001 Society of Chemical Industry [source]


Differential gene expression in LPS/IFN, activated microglia and macrophages: in vitro versus in vivo

JOURNAL OF NEUROCHEMISTRY, Issue 2009
Christoph D. Schmid
Abstract Two different macrophage populations contribute to CNS neuroinflammation: CNS-resident microglia and CNS-infiltrating peripheral macrophages. Markers distinguishing these two populations in tissue sections have not been identified. Therefore, we compared gene expression between LPS (lipopolysaccharide)/interferon (IFN),-treated microglia from neonatal mixed glial cultures and similarly treated peritoneal macrophages. Fifteen molecules were identified by quantative PCR (qPCR) as being enriched from 2-fold to 250-fold in cultured neonatal microglia when compared with peritoneal macrophages. Only three of these molecules (C1qA, Trem2, and CXCL14) were found by qPCR to be also enriched in adult microglia isolated from LPS/IFN,-injected CNS when compared with infiltrating peripheral macrophages from the same CNS. The discrepancy between the in vitro and in vivo qPCR data sets was primarily because of induced expression of the ,microglial' molecules (such as the tolerance associated transcript, Tmem176b) in CNS-infiltrating macrophages. Bioinformatic analysis of the ,19000 mRNAs detected by TOGA gene profiling confirmed that LPS/IFN,-activated microglia isolated from adult CNS displayed greater similarity in total gene expression to CNS-infiltrating macrophages than to microglia isolated from unmanipulated healthy adult CNS. In situ hybridization analysis revealed that nearly all microglia expressed high levels of C1qA, while subsets of microglia expressed Trem2 and CXCL14. Expression of C1qA and Trem2 was limited to microglia, while large numbers of GABA+ neurons expressed CXCL14. These data suggest that (i) CNS-resident microglia are heterogeneous and thus a universal microglia-specific marker may not exist; (ii) the CNS micro-environment plays significant roles in determining the phenotypes of both CNS-resident microglia and CNS-infiltrating macrophages; (iii) the CNS microenvironment may contribute to immune privilege by inducing macrophage expression of anti-inflammatory molecules. [source]


1,1-bis(3,-indolyl)-1-(p -methoxyphenyl)methane activates Nur77-independent proapoptotic responses in colon cancer cells

MOLECULAR CARCINOGENESIS, Issue 4 2008
Sung Dae Cho
Abstract 1,1-Bis(3,-indolyl)-1-(p -methoxyphenyl)methane (DIM-C-pPhOCH3) is a methylene-substituted diindolylmethane (C-DIM) analog that activates the orphan receptor nerve growth factor-induced-B, (NGFI-B,, Nur77). RNA interference studies with small inhibitory RNA for Nur77 demonstrate that DIM-C-pPhOCH3 induces Nur77-dependent and -independent apoptosis, and this study has focused on delineating the Nur77-independent proapoptotic pathways induced by the C-DIM analog. DIM-C-pPhOCH3 induced caspase-dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl-2 ratios and release of cytochrome c into the cytosol. DIM-C-pPhOCH3 also induced phosphatidylinositol-3-kinase-dependent activation of early growth response gene-1 which, in turn, induced expression of the proapoptotic nonsteroidal anti-inflammatory drug-activated gene-1 (NAG1) in RKO and SW480 colon cancer cells. Moreover, DIM-C-pPhOCH3 also induced NAG-1 expression in colon tumors in athymic nude mice bearing RKO cells as xenografts. DIM-C-pPhOCH3 also activated the extrinsic apoptosis pathway through increased phosphorylation of c- jun N-terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5). Thus, the effectiveness of DIM-C-pPhOCH3 as a tumor growth inhibitor is through activation of Nur77-dependent and -independent pathways. © 2007 Wiley-Liss, Inc. [source]


An anti-inflammatory oligopeptide produced by Entamoeba histolytica down-regulates the expression of pro-inflammatory chemokines

PARASITE IMMUNOLOGY, Issue 10 2003
Dolores Utrera-Barillas
SUMMARY Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with anti-inflammatory properties that, among others, inhibits the in vitro and in vivo locomotion of human monocytes, sparing polymorphonuclear leucocytes from this effect [hence the name originally given: Monocyte Locomotion Inhibitory Factor (MLIF)]. A synthetic construct of this peptide displays the same effects as the native material. We now added MLIF to resting and PMA-stimulated cells of a human monocyte cell line and measured the effect upon mRNA and protein expression of pro-inflammatory chemokines (RANTES, IP-10, MIP-1,, MIP-1,, MCP-1, IL-8, I-309 and lymphotactin) and the shared CC receptor repertoire. The constitutive expression of these chemokines and the CC receptors was unaffected, whereas induced expression of MIP-1,, MIP-1,, and I-309, and that of the CCR1 receptor , all involved in monocyte chemotaxis , was significantly inhibited by MLIF. This suggests that the inhibition of monocyte functions by MLIF may not only be exerted directly on these cells, but also , and perhaps foremost , through a conglomerate down-regulation of endogenous pro-inflammatory chemokines. [source]


S -Allyl- L -Cysteine Sulfoxide Inhibits Tumor Necrosis Factor-Alpha Induced Monocyte Adhesion and Intercellular Cell Adhesion Molecule-1 Expression in Human Umbilical Vein Endothelial Cells

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2010
Chai Hui
Abstract Garlic and its water-soluble allyl sulfur-containing compound, S -Allyl- L -cysteine Sulfoxide (ACSO), have shown antioxidant and anti-inflammatory activities, inhibiting the development of atherosclerosis. However, little is known about the mechanism(s) underlying the therapeutic effect of ACSO in inhibiting the formation of atherosclerostic lesion. This study aimed to investigate whether ACSO could modulate tumor necrosis factor-alpha (TNF-,)-induced expression of intercellular cell adhesion molecule-1, monocyte adhesion and TNF-,-mediated signaling in human umbilical vein endothelial cells. While TNF-, promoted the intercellular cell adhesion molecule-1 mRNA transcription in a dose- and time-dependent manner, ACSO treatment significantly reduced the levels of TNF-,-induced intercellular cell adhesion molecule-1 mRNA transcripts (P < 0.01). Furthermore, ACSO dramatically inhibited TNF-, triggered adhesion of THP-1 monocytes to endothelial cells and porcine coronary artery rings. Moreover, ACSO mitigated TNF-, induced depolarization of mitochondrial membrane potential and overproduction of superoxide anion, associated with the inhibition of NOX4, a subunit of nicotinamide adenine dinucleotide phosphate-oxidase, mRNA transcription. In addition, ACSO also inhibited TNF-,-induced phosphorylation of JNK, ERK1/2 and I,B, but not p38. Apparently, ACSO inhibited proinflammatory cytokine-induced adhesion of monocytes to endothelial cells by inhibiting the mitogen-activated protein kinase signaling and related intercellular cell adhesion molecule-1 expression, maintaining mitochondrial membrane potential, and suppressing the overproduction of superoxide anion in endothelial cells. Therefore, our findings may provide new insights into ACSO on controlling TNF-,-mediated inflammation and vascular disease. Anat Rec, 2010. © 2010 Wiley-Liss, Inc. [source]


ABA activates ADPR cyclase and cADPR induces a subset of ABA-responsive genes in Arabidopsis

THE PLANT JOURNAL, Issue 3 2004
Juan-Pablo Sánchez
Summary Cyclic ADP-ribose (cADPR) was previously shown to activate transient expression of two abscisic acid (ABA)-responsive genes in tomato cells. Here, we show that the activity of the enzyme responsible for cADPR synthesis, ADP-ribosyl (ADPR) cyclase, is rapidly induced by ABA in both wild-type (WT) and abi1-1 mutant Arabidopsis plants in the absence of protein synthesis. Furthermore, in transgenic Arabidopsis plants, induced expression of the Aplysia ADPR cyclase gene resulted in an increase in ADPR cyclase activity and cADPR levels, as well as elevated expression of ABA-responsive genes KIN2, RD22, RD29a, and COR47 (although to a lesser extent than after ABA induction). Genome-wide profiling indicated that about 28% of all ABA-responsive genes in Arabidopsis are similarly up- and downregulated by cADPR and contributed to the identification of new ABA-responsive genes. Our results suggest that activation of ADPR cyclase is an early ABA-signaling event partially insensitive to the abi1-1 mutation and that an increase in cADPR plays an important role in downstream molecular and physiological ABA responses. [source]


5,-dihydrotestosterone inhibits 1,,25-dihydroxyvitamin D3 -induced expression of CYP24 in human prostate cancer cells

THE PROSTATE, Issue 3 2005
Yan-Ru Lou
Abstract BACKGROUND A cross-talk between 1,,25-dihydroxyvitamin D3 [1,,25-(OH)2D3] and 5,-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. METHODS The expression of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1,,25-(OH)2D3 was measured using a radioreceptor assay. RESULTS Real-time RT-PCR showed that DHT at 1,100 nM significantly inhibited 1,,25-(OH)2D3 -induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1,,25-(OH)2D3 was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. CONCLUSIONS We demonstrated, for the first time, that DHT reduces the ability of 1,,25-(OH)2D3 to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact. © 2004 Wiley-Liss, Inc. [source]


Trex-1 deficiency in rheumatoid arthritis synovial fibroblasts

ARTHRITIS & RHEUMATISM, Issue 9 2010
Michel Neidhart
Objective To explore whether the increased expression of long interspersed nuclear element 1 (LINE-1; L1) messenger RNA (mRNA) and protein in rheumatoid arthritis synovial fibroblasts (RASFs) is associated with decreased expression of Trex-1, an exonuclease involved in the metabolization of L1 DNA:RNA hybrids. Methods Chromatin immunoprecipitation was used to detect L1-related p40 protein (L1-ORF1p) binding sequences in RASFs. Luciferase activity was measured in the synovial fibroblasts following cotransfection of the episomal plasmid with pJM105 expressing L1-ORF1p and pGL3-TS3 carrying the target sequence for L1-ORF1p. This luciferase reporter assay was used to compare the activity between RASFs and osteoarthritis synovial fibroblasts (OASFs) and to assess correlations of luciferase activity with the expression of Trex-1 measured by flow cytometry. The expression of Trex-1 mRNA and protein was also compared using real-time polymerase chain reaction, immunohistochemistry, and Western blot analyses. The role of Trex-1 in the L1-ORF1p,mediated luciferase activity assay was studied using interfering RNAs (iRNA) and a Trex-1 expression vector. Results Increased luciferase activity occurred after cotransfection of synovial fibroblasts with pJM105 and pGL3-TS3. L1-ORF1p activity was increased in RASFs as compared with OASFs, and this was correlated inversely with the expression of Trex-1. Levels of Trex-1 mRNA and protein were lower in RASFs than in OASFs. After transfection of the L1 expression plasmid, Trex-1 mRNA levels increased in OASFs, but not in RASFs. The addition of iRNA against Trex-1, however, resulted in an enhancement of L1-ORF1p activity in OASFs to the levels measured in RASFs. Overexpression of Trex-1 inhibited 5-azacytidine,induced expression of p38, MAPK, a gene carrying the TS3 sequence. Conclusion The deficiency of Trex-1 in RASFs allows a longer half-life of gene products encoded by active endogenous L1 retrotransposons. This pathway may play a role in diseases in which the cells exhibit a "spontaneous" aggressive behavior. [source]


MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes

ARTHRITIS & RHEUMATISM, Issue 5 2010
Nahid Akhtar
Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin-1, (IL-1,) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR),based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription,PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-,B was evaluated using specific inhibitors. Results In IL-1,,stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3,-untranslated region (3,-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3,-UTR of human MMP-13 mRNA and inhibited the IL-1,,induced expression of MMP-13 protein in chondrocytes. NF-,B and MAPK activation down-regulated the expression of miR-27b. Conclusion Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1,,induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes. [source]


Involvement of MAPKs and NF-,B in tumor necrosis factor ,,induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts

ARTHRITIS & RHEUMATISM, Issue 1 2010
Shue-Fen Luo
Objective To investigate the roles of MAPKs and NF-,B in tumor necrosis factor , (TNF,),induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). Methods Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-,B in TNF,-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase,polymerase chain reaction, and gene promoter assay. NF-,B translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results TNF,-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-,B were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-,B (helenalin) or by transfection with their respective shRNA. TNF,-stimulated translocation of NF-,B into the nucleus and NF-,B promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF, in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNF, or by anti,VCAM-1 antibody before the addition of lymphocytes. Conclusion In RASFs, TNF,-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-,B pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches. [source]


Tumor necrosis factor ,,induced interleukin-32 is positively regulated via the Syk/protein kinase C,/JNK pathway in rheumatoid synovial fibroblasts

ARTHRITIS & RHEUMATISM, Issue 3 2009
Se Hwan Mun
Objective Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. Methods FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase,polymerase chain reaction, and small interfering RNA (siRNA) transfection. Results IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor , (TNF,). TNF,-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase C, (PKC,), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKC, mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKC,. Conclusion The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKC,-mediated signaling events. [source]


IK cytokine ameliorates the progression of lupus nephritis in MRL/lpr mice

ARTHRITIS & RHEUMATISM, Issue 11 2006
Masatake Muraoka
Objective IK cytokine has been isolated as a factor that inhibits interferon-, (IFN,),induced expression of class II major histocompatibility complex (MHC) antigens. Aberrant expression of class II MHC antigens has reportedly been recognized in the target organs of autoimmune diseases and been associated with disease activity. In this study, we investigated whether IK cytokine can ameliorate the progression of lupus nephritis in MRL/lpr mice. Methods A truncated IK analog was prepared and transfected into a nonmetastatic fibroblastoid cell line, and then injected subcutaneously into MRL/lpr mice at ages 8 weeks (before the onset of lupus nephritis) and 12 weeks (at the early stage of the disease). Results An IK cytokine, when it was translated from methionine at position 316, acted as a secretory protein. This truncated IK cytokine (tIK) reduced IFN,-induced class II MHC expression in various cells through decreased expression of class II MHC transcription activator. Treatment of MRL/lpr mice with tIK significantly reduced renal damage as compared with control mice. A significant decrease in macrophage and T cell infiltration was found in the kidneys of tIK-treated mice, resulting in decreased production of IFN, and interleukin-2. Mice treated with tIK also showed significant reduction of anti-DNA antibodies and circulating immune complexes. A specific reduction of class II MHC expression was observed on B cells and monocytes as well as in the kidney. Conclusion We prepared a potent IK analog and demonstrated its ability to ameliorate the progression of lupus nephritis. This agent may therefore provide a new therapeutic approach for lupus nephritis. [source]


Rationally engineered biotransformation of p -nitrophenol

BIOTECHNOLOGY PROGRESS, Issue 3 2010
Matthew de la Peña Mattozzi
Abstract An operon encoding enzymes responsible for degradation of the EPA priority contaminant para -nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad-host-range, BioBricks-compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of ,-ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]