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At least 325 records · Page 18Linked to original sources
PubMed2002

Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.

Chemical Precipitation↗
PubMed2004

Increase of myocardial salvage and left ventricular function recovery with intracoronary abciximab downstream of the coronary occlusion in patients with acute myocardial infarction treated with primary coronary intervention.

In this prospective randomized trial on patients with acute myocardial infarction (AMI) treated with primary percutaneous coronary intervention (PCI), we hypothesized that abciximab administered intracoronarily, downstream of the coronary occlusion, leads to a greater degree of myocardial salvage and better left ventricular function recovery compared with the usual abciximab administration. Forty-five consecutive patients with first AMI and infarct-related artery TIMI flow 0-1 undergoing primary PCI were enrolled. Twenty-two patients were randomly assigned to the intracoronary treatment and 23 to the usual treatment. The initial perfusion defect, final infarct size, myocardial salvage, salvage index, and left ventricular function recovery were assessed by serial scintigraphic scans performed at admission and 7 days and 1 month after PCI. Angiographic myocardial blush grade, corrected TIMI frame count, and electrocardiographic ST segment elevation reduction were also assessed as markers of myocardial reperfusion. Final infarct size was significantly smaller (P = 0.043) and salvage index significantly higher (P = 0.003) in the intracoronary treatment group as a result of a greater degree of myocardial salvage (P = 0.0001). The increase of left ventricular ejection fraction at 1 month was significantly higher in the intracoronary treatment patients (P = 0.013). The markers of myocardial reperfusion were also significantly better in the intracoronary treatment group. In patients with AMI and occluded infarct-related artery treated with primary PCI, intracoronary abciximab given just before PCI downstream of the occlusion is associated to a greater degree of myocardial salvage than the usual abciximab protocol. This benefit is mainly related to a substantial reduction in final infarct size, which leads to an improvement in left ventricular ejection fraction.

Abciximab↗
PubMed2003

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping.

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.

Animals↗
PubMed1995

Protein kinase C is not a downstream effector of p21ras in activated T cells.

The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin-2 (IL-2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF-AT and AP-1 transcriptional activity and in the expression of IL-2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF-kappa B transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL-2 during T cell activation.

DNA-Binding Proteins↗
PubMed2003

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase, respectively.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

Antigens, CD19↗
PubMed2005

Adenosine and cAMP are potent inhibitors of the NF-kappa B pathway downstream of immunoreceptors.

Anergic B lymphocytes exert compromised signal transduction towards the activation of NF-kappa B in response to B cell antigen receptor (BCR) triggering, whereas activation of the ERK pathway appears normal. How this differential down-regulation of the NF-kappa B pathway is regulated remains still elusive. Here, we demonstrate that stimuli known to enhance 3',5'-cyclic adenosine monophosphate (cAMP) are capable of selectively suppressing the activation both of NF-kappa B downstream of the BCR and Toll-like receptor 4 in splenic B lymphocytes and of the high-affinity receptor for IgE in BM-derived mast cells. This suppression is accomplished by blocking phosphorylation and subsequent degradation of the inhibitor of NF-kappa B. A cAMP-dependent protein kinase (PKA) inhibitor reverses this suppressive effect, indicating that PKA is a downstream effector of cAMP in this process. Importantly, not only drugs that artificially elevate intracellular cAMP levels, but also the nucleoside adenosine, which is known to be a mediator of cellular distress, inhibit the NF-kappa B pathway. This suggests that adenosine-mediated signals represent an important step in the molecular decision process controlling inflammation versus anergic immune responses.

1-Methyl-3-isobutylxanthine↗
PubMed2005

The downstream financial effect of hepatology.

As a more consultative but less procedurally oriented specialty, Hepatology has been considered a financial liability in some academic centers. However, no actual data exist on the relative contribution of a Hepatology practice. The purpose of this study was to evaluate the direct and indirect (i.e., downstream effect) charges generated by a Hepatology section in comparison with a Gastroenterology section. Using a computerized database, retrospective cohorts of new outpatient consultations and initial admissions seen by the Hepatology and Gastroenterology sections over a 3-month period were created. The cohorts were followed for 12 months. Charges generated directly to the section (direct charges) and to the hospital system (indirect charges) were calculated. Each cohort consisted of 179 patients. The Hepatology patients generated 5,851,463 dollars in overall charges for the hospital, compared with 2,273,339 dollars for the Gastroenterology cohort. Only 3.6% of the Hepatology charges were direct, compared with 15.9% of the Gastroenterology charges. For every 1 dollar billed by Hepatology, the hospital system generated an additional 26.95 dollars in charges (51.03 dollars for the orthotopic liver transplantation patients, and 14.26 dollars for the non-orthotopic liver transplantation patients). For every 1 dollar billed by Gastroenterology, the hospital system generated an additional 5.31 dollars in charges. Similar inpatient collection rates were seen between the two groups (27.7% for hepatology and 33.6% for gastroenterology). In conclusion, although Hepatology generates only a small amount of direct charges, it accounts for a very substantial amount of indirect or downstream billing for an academic medical center. This study validates the importance of a hospital's support for a Hepatology section, especially in a center performing orthotopic liver transplantation.

Academic Medical Centers↗
PubMed2002

Dysregulation of phosphatidylinositol 3-kinase and downstream effectors in human breast cancer.

Phosphatidylinositol 3-kinase (PI3-K) is a growth factor-activated transforming lipid (and protein) kinase, involved in cell motility and invasion, that has multiple effectors. Relatively little is known about its expression and enzymatic activity in human breast cancer. Since growth factor receptors are amplified in breast cancer, and the tumor suppressor PTEN may be mutated in human breast cancer, it was hypothesized that PI3-K and its downstream effectors would be activated in this disease. In 11 resected tumors analyzed for expression of this kinase, a mean 3-fold increase in protein expression was observed over the corresponding adjacent control tissue. Using an in vitro lipid kinase assay of the immunoprecipitated PI3-K protein, a greater than 2-fold increase in activation was observed. These changes were observed in the absence of an activation of either protein kinase B (PKB, akt1) or p70 S6 kinase (p70 S6K). However, p21-activated kinase (Pak), p38 mitogen-activated protein kinase (p38 MAPK) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK 2) were all overexpressed and demonstrated increased enzyme activity. It may be concluded that aberrant mitogenic signaling in human breast cancer in vivo involves Pak, p38 MAPK and MAPKAPK2 downstream of PI3-K, but neither of PKB or p70 S6K. It is proposed that this pathway may serve as a useful targeting nexus for investigation of small molecule inhibitors in human breast cancer.

Breast Neoplasms↗
PubMed2005

Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Iressa, ZD1839) in chemotherapy-resistant non-small cell lung cancer.

Gefitinib has shown meaningful antitumor activity with tolerable toxicity in chemotherapy-refractory NSCLC in previous studies. Moreover, EGFR expression failed to show a correlation with response. In an attempt to identify predictive markers of response, we have investigated the tumoral expression of key signaling molecules of EGFR (EGFR, p-EGFR, p-Akt, p-Erk, p-STAT3) by immunohistochemistry and analyzed their correlations with response. Of 65 patients who received gefitinib (250 mg/day) for chemotherapy-refractory NSCLC, there were 14 partial responses (21.5%), 21 stable diseases (32.3%) and 21 progressive diseases (32.3%). Median durations of overall survival and time to progression were 6.7 months and 2.8 months, respectively. Immunohistochemistry was performed in 34 patients with evaluable tissue specimens. EGFR was overexpressed (2+ or 3+) in 32.4% and p-EGFR was positive in 26.5%. The expressions of p-Akt, p-Erk and p-STAT3 were positive (1+ or 2+) in 50%, 38.2% and 79.4%, respectively. The EGFR expression was not correlated with p-EGFR or the downstream molecules. EGFR or p-EGFR status did not correlate with response. Positive expression of p-Erk was significantly associated with poor response (38.1% in -, 14.3% in 1+, 0% in 2+; p = 0.046). Furthermore, tumors with positive p-Akt and negative p-Erk nuclear expression exhibited the best response (60%), whereas there was no response in the opposite [p-Akt (-), p-Erk (+)] cases. Intense nuclear staining of p-Akt (2+) was associated with prolonged TTP (HR 0.25, 95% confidence interval [CI] 0.08-0.79, p = 0.018) and OS (HR 0.16, 95% CI 0.04-0.62, p = 0.008). These results support the assumption that gefitinib responsiveness might be predicted by activated EGFR downstream molecules such as p-Akt and p-Erk.

Adult↗
PubMed1988

Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

To characterize cis-acting elements controlling the expression of T cell receptor beta-chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta-chain with different extensions of 5' and 3' flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements.

Animals↗
PubMed1992

The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.

In the yeast Saccharomyces cerevisiae the G-protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant-negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G-protein mediated signal transduction, the activation of a protein kinase through G beta gamma.

Amino Acid Sequence↗
PubMed1995

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

Amino Acid Sequence↗
PubMed2006

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK.

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.

Animals↗
PubMed2007

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation.

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.

Animals↗
PubMed1999

Cbl functions downstream of Src kinases in Fc gamma RI signaling in primary human macrophages.

Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated in response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck. A few reports describe Cbl interactions in primary human hematopoietic cells. We show evidence that Cbl participates in signaling initiated by Fc gammaRI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway. Fc gammaRI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein. Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38-kDa band identified as the CrkL adaptor protein. CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after Fc gammaRI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after Fc gammaRI stimulation and was associated with both Cbl and Grb2. Grb2 and Cbl binding to SLP-76 were inducible after Fc gammaRI stimulation of the macrophages. Nck was inducibly bound to Cbl after Fc gammaRI stimulation, whereas Grb2 was constitutively associated with it. Shc was also inducibly tyrosine phosphorylated and bound to Grb2 after Fc gammaRI stimulation of the macrophages. PP1, a specific inhibitor of Src kinases, inhibited the Fc gammaRI-induced respiratory burst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL. These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and the association of Cbl with CrkL, SLP-76, and Nck in Fc gammaRI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Nicotinic alpha 7 receptors: synaptic options and downstream signaling in neurons.

Nicotinic receptors are cation-ion selective ligand-gated ion channels that are expressed throughout the nervous system. Most have significant calcium permeabilities, enabling them to regulate calcium-dependent events. One of the most abundant is a species composed of the alpha 7 gene product and having a relative calcium permeability equivalent to that of NMDA receptors. The alpha 7-containing receptors can be found presynaptically where they modulate transmitter release, and postsynaptically where they generate excitatory responses. They can also be found in perisynaptic locations where they modulate other inputs to the neuron and can activate a variety of downstream signaling pathways. The effects the receptors produce depend critically on the sites at which they are clustered. Instructive preparations for examining alpha 7-containing receptors are the rat hippocampus, where they are thought to play a modulatory role, and the chick ciliary ganglion, where they participate in throughput transmission as well as regulatory signaling. Relatively high levels of alpha 7-containing receptors are found in the two preparations, and the receptors display a variety of synaptic options and functions in the two cases. Progress is starting to be made in understanding the mechanisms responsible for localizing the receptors at specific sites and in identifying components tethered in the vicinity of the receptors that may facilitate signal transduction and downstream signaling.

Animals↗
PubMed1995

Reversion of v-H-ras-transformed NIH 3T3 cells by apigenin through inhibiting mitogen activated protein kinase and its downstream oncogenes.

Apigenin, a plant flavonoid, induced the reversion of transformed phenotypes of v-H-ras-transformed NIH 3T3 cells at a quite low concentration of 12.5 microM. In the present study, we have examined the components of this Ras-mediated signaling transduction to determine whether they were involved in the apigenin-induced reversion process. Interestingly, the consitutively activated mitogen activated protein kinase (MAPK) in the ras transformant was inhibited significantly and rapidly by 25 microM apigenin within 30 min, and this reduction continued for more than 4 h. Corroborating these observations, expression of the downstream oncogenes c-jun and c-fos was also dramatically reduced during the first 4 h of treatment. We found that the levels of ras protein and mRNA were not affected by 24 h of treatment with apigenin. These findings indicate that apigenin-induced reversion of v-H-ras-transformed NIH 3T3 cells may occur by inhibiting MAPK activity and its downstream oncogenes rather than by affecting the expression of the ras gene.

3T3 Cells↗
PubMed2001

Serum after partial hepatectomy stimulates iNOS gene transcription via downstream NF-kappa B site.

It has been known that the expression of inducible nitric oxide synthase (iNOS) is up-regulated during hepatic regeneration. The present study characterized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) in vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-alpha (TNF-alpha) antibody in BNL CL.2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-kappa B-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of NF-kappa B complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcriptional activation of iNOS promoter by post-PH serum. Furthermore, site-directed mutational analysis of the two NF-kappa B sites individually or in combination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-kappa B site, but not by upstream NF-kappa B site. Taken together, these results suggest that the downstream NF-kappa B site acts as an essential component for the iNOS expression by post-PH serum during hepatic regeneration.

Animals↗