SEARCH · PubMed Health

Results for “virtual protein profiling”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed2004

Loss of cyclin E requirement in cell growth of an oral squamous cell carcinoma cell line implies deregulation of its downstream pathway.

Cyclin E and Cdk2 have been shown to play an important role in G1/S transition of the cell cycle. Two E-type cyclins (E1 and E2) have been identified to date and share functionally similarities. Upregulation of these cyclins has been observed frequently in human cancers. We examined the expression profile of cyclin E1 and E2 in cell lines derived from human oral squamous cell carcinoma (SCC), and found that the expression of cyclin E1 protein was hardly detected in HSC-2 cells. Although cyclin E2 was abundantly expressed, histone H1 kinase activities of both E-type cyclins were virtually undetectable in this cell line. Inhibition of cyclin E1, but not that of E2, by using vectors expressing antisense-oriented their cDNAs induced drastic growth suppression on HOC313 cells that express both E-type cyclins. Inhibition of neither cyclin E1 nor E2 suppressed the growth of HSC-2 cells, and compensatory elevation of cyclin E1 was not evident in cyclin E2-inhibited HSC-2 cells. In contrast, HSC-2 cells expressed cyclin D1 and hyperphosphorylated forms of Rb family proteins, and were arrested in G1 by overexpression of p16(INK4), a specific inhibitor against D-type cyclin activity. These results indicate that HSC-2 cells lost proper growth control specifically mediated by cyclin E and suggest that deregulation of its downstream pathway may contribute to tumorigenesis of oral SCC.

Cell Division↗

PubMed2006

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin.

Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.

Amino Acid Substitution↗

PubMed1982

Adenosine receptor agonists: binding and adenylate cyclase stimulation in rat liver plasma membranes.

N6-Cyclohexyl[3H]adenosine([3H]CHA,[3H]adenosine, and 5'N-ethylcarboxamide[3H]adenosine ([3H]NECA), potent agonists in adenosine-responsive cellular systems, have been used to identify adenosine binding sites in rat liver plasma membranes. Endogenous ligands were removed by prior dialysis of the membranes. Specific binding of the ligands tested was characterized by rapid forward and reverse kinetics and heterogeneity as indicated by curvilinear Scatchard plots. The KD in the high affinity range was 80 nM for [3H]adenosine, 84 nM for [3H]NECA, and 168 nM for [3H]CHA; the respective binding capacities of 1.19, 1.03, and 1.05 pmol/mg protein were of virtually the same magnitude, suggesting labeling of identical sites. However, all ligands also displayed binding to large numbers of low affinity sites. This high level of apparently non-receptor binding markedly influenced the adenosine structure-activity profile of [3H]CHA displacement, which differs with pharmacological findings. - NECA and CHA stimulated hepatic adenylate cyclase with an apparent ED50 of 60 and 580 nM, respectively; adenosine was stimulatory at a concentration range from 0.1 - 2.0 microM, but inhibitory at higher concentrations. Hence, estimation of the true ED50 was not possible. Because the KD of high affinity binding and the ED50 of the biological effect of NECA and CHA are in the same range, it may be reasonable to assume that the high affinity sites represent adenosine receptors, recently classified as Ra-site receptors.

Adenosine↗

PubMed1990

C595--a monoclonal antibody against the protein core of human urinary epithelial mucin commonly expressed in breast carcinomas.

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.

Antibodies, Monoclonal↗

PubMed1990

Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sar9,Met(O2)11]-substance P.

The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11]-substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H]-[Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes.

Animals↗

PubMed2005

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats.

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.

Aging↗

PubMed2004

Global analysis of HuR-regulated gene expression in colon cancer systems of reducing complexity.

HuR, a protein that binds to target mRNAs and can enhance their stability and translation, is increasingly recognized as a pivotal regulator of gene expression during cell division and tumorigenesis. We sought to identify collections of HuR-regulated mRNAs in colon cancer cells by systematic, cDNA array-based assessment of gene expression in three systems of varying complexity. First, comparison of gene expression profiles among tumors with different HuR abundance revealed highly divergent gene expression patterns, and virtually no changes in previously reported HuR target mRNAs. Assessment of gene expression patterns in a second system of reduced complexity, cultured colon cancer cells expressing different HuR levels, rendered more conserved sets of HuR-regulated mRNAs. However, the definitive identification of direct HuR target mRNAs required a third system of still lower complexity, wherein HuR-RNA complexes immunoprecipitated from colon cancer cells were subject to cDNA array hybridization to elucidate the endogenous HuR-bound mRNAs. Comparison of the transcript sets identified in each system revealed a strikingly limited overlap in HuR-regulated mRNAs. The data derived from this systematic analysis of HuR-regulated genes highlight the value of low-complexity, biochemical characterization of protein-RNA interactions. More importantly, however, the data underscore the broad usefulness of integrated approaches comprising systems of low complexity (protein-nucleic acid) and high complexity (cells, tumors) to comprehensively elucidate the gene regulatory events that underlie biological processes.

Animals↗

PubMed2002

Agonist-induced translocation of the kinin B(1) receptor to caveolae-related rafts.

The kallikrein-kinin system, activated during inflammatory conditions and the regulation of specific cardiovascular and renal functions, includes two G protein-coupled receptors for bradykinin (BK)-related peptides. The B(1) receptor (B(1)R) subtype is not believed to undergo agonist-induced phosphorylation and endocytosis. A conjugate made of the rabbit B(1)R fused with the yellow variant of green fluorescent protein (YFP) was expressed in mammalian cells. In COS-1 or human embryonic kidney (HEK) 293 cells, the construction exhibited a nanomolar affinity for the agonist radioligand [(3)H]Lys-des-Arg(9)-BK or the antagonist ligand [(3)H]Lys-[Leu(8)]des-Arg(9)-BK and a pharmacological profile virtually identical to that of wild-type B(1)R. Lys-des-Arg(9)-BK stimulation of HEK 293 cells stably expressing B(1)R-YFP but not stimulation of untransfected cells released [(3)H]arachidonate in a phospholipase A(2) assay. B(1)R-YFP was visualized as a continuous labeling of the plasma membranes in stably transfected HEK 293 cells (confocal microscopy). Addition of Lys-des-Arg(9)-BK (1-100 nM) rapidly concentrated the receptor-associated fluorescence into multiple aggregates that remained associated with the plasma membrane (no significant internalization) and colocalized with caveolin-1. This reaction was slowly reversible upon agonist washing at 37 degrees C and prevented pretreatment with a B(1)R antagonist. beta-Cyclodextrin treatment, which extracts cholesterol from membranes and disrupts caveolae-related rafts, prevented agonist-induced redistribution of B(1)R-YFP but not the PLA(2) activation mediated by this receptor. The agonist radioligand copurified with caveolin-1 to a greater extent than the tritiated antagonist in buoyant fractions of HEK 293 cells treated with the ligands. Agonist-induced cellular translocation of the kinin B(1)R to caveolae-related rafts without endocytosis is a novel variation on the theme of G protein-coupled receptor adaptation.

Animals↗

PubMed1988

Modes of DNA cleavage by the EcoRV restriction endonuclease.

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.

DNA↗

PubMed1982

Intracellular nucleocapsid RNA of mumps virus.

Intracellular nucleocapsids, virtually free of host proteins and nucleic acids, were purified from cells acutely infected with several strains of mumps virus. VIral RNA obtained from the nucleocapsids was predominantly a single 50S species, with 28S RNA occurring as a secondary peak in the RNA profile. Nucleocapsids yielded approx. 9 micrograms 50S RNA per 10(9) infected cells. The 50S RNA was partially self-complementary, indicating that mumps viral nucleocapsids include full-length plus (+) sense antigenomes. Self-annealing and competition hybridization assays with 50S RNA suggest that 28S RNA includes duplexes consisting of full-length (+) and minus (-) sense 50S RNA. Properties of the viral RNA did not vary with the method of RNA isolation, time post-infection at which nucleocapsids were harvested, nor the strain of mumps virus.

Animals↗

PubMed2007

Gene expression and protein length influence codon usage and rates of sequence evolution in Populus tremula.

Codon bias is generally thought to be determined by a balance between mutation, genetic drift, and natural selection on translational efficiency. However, natural selection on codon usage is considered to be a weak evolutionary force and selection on codon usage is expected to be strongest in species with large effective population sizes. In this paper, I study associations between codon usage, gene expression, and molecular evolution at synonymous and nonsynonymous sites in the long-lived, woody perennial plant Populus tremula (Salicaceae). Using expression data for 558 genes derived from expressed sequence tags (EST) libraries from 19 different tissues and developmental stages, I study how gene expression levels within single tissues as well as across tissues affect codon usage and rates sequence evolution at synonymous and nonsynonymous sites. I show that gene expression have direct effects on both codon usage and the level of selective constraint of proteins in P. tremula, although in different ways. Codon usage genes is primarily determined by how highly expressed a genes is, whereas rates of sequence evolution are primarily determined by how widely expressed genes are. In addition to the effects of gene expression, protein length appear to be an important factor influencing virtually all aspects of molecular evolution in P. tremula.

Codon↗

PubMed1984

Capacity of lipoproteins to act as substrates for lecithin:cholesterol acyltransferase. Enhancement by pre-incubation with an artificial triacylglycerol emulsion.

Previous studies have shown that high concentrations of very low density lipoproteins (VLDL) stimulate the formation of cholesteryl esters in human plasma, possibly by acting as recipients of cholesteryl esters transferred from high density lipoproteins (HDL). To gain further insight into this phenomenon, experiments were performed to determine whether the triacylglycerol emulsion Intralipid, which acts as an artificial recipient of HDL cholesteryl esters, has an effect on cholesterol esterification similar to that of VLDL. Intralipid, in contrast to VLDL, is devoid of apolipoproteins which may stimulate cholesterol esterification. Human plasma, which had previously been depleted of VLDL, was pre-incubated in the presence or absence of Intralipid. After pre-incubation, the Intralipid was removed and rates of cholesterol esterification were measured in subsequent incubations of the Intralipid-depleted fractions. The presence of Intralipid during the pre-incubation had a marked stimulatory effect on cholesterol esterification, comparable to that of VLDL in earlier studies. The pre-incubation with Intralipid also markedly reduced the cholesteryl ester content, and increased the triacylglycerol content, of the HDL. These changes in composition coincided with two changes in the elution profile of HDL after density-gradient ultracentrifugation which were (i) a reduction in the density of particles in the HDL3 subfraction, which virtually disappeared as an identifiable peak, and (ii) the emergence of a discrete population of very dense lipoproteins consisting primarily of protein and phospholipid. Since all these changes were related to redistributions of lipids, the results highlight the importance of lipid transfers in the regulation of plasma cholesterol esterification.

Cholesterol Esters↗

PubMed1987

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model.

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.

Animals↗

PubMed2004

The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity.

A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants. The structure of the 28 kDa protein, which had been envisaged to represent a precursor, or fusion product of the two more specialized, common chloroplast thioredoxins f and m has now been determined by amino acid sequencing. Although it exhibits virtually all the properties and enzyme-modulating activities of a thioredoxin proper this algal protein, surprisingly, does not belong to the thioredoxin family of small redox proteins but is identical with OEE (oxygen evolving enhancer) protein 1, an auxiliary component of the photosystem II manganese cluster. Extracts of Chlorella vulgaris and Chlamydomonas reinhardtii also contain heat-stable protein fractions of 23-26 kDa capable of specifically stimulating chloroplast fructosebisphosphatase in vitro. In contrast, OEE protein 1 from spinach is not able to modulate FbPase or NADP malate dehydrogenase from spinach chloroplasts. A dual function of the OEE protein in algal photosynthesis is envisaged.

Algal Proteins↗

PubMed2006

Mutations in the gene encoding 3-hydroxyisobutyryl-CoA hydrolase results in progressive infantile neurodegeneration.

Only a single patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency has been described in the literature, and the molecular basis of this inborn error of valine catabolism has remained unknown until now. Here, we present a second patient with 3-hydroxyisobutyryl-CoA hydrolase deficiency, who was identified through blood spot acylcarnitine analysis showing persistently increased levels of hydroxy-C(4)-carnitine. Both patients manifested hypotonia, poor feeding, motor delay, and subsequent neurological regression in infancy. Additional features in the newly identified patient included episodes of ketoacidosis and Leigh-like changes in the basal ganglia on a magnetic resonance imaging scan. In cultured skin fibroblasts from both patients, the 3-hydroxyisobutyryl-CoA hydrolase activity was deficient, and virtually no 3-hydroxyisobutyryl-CoA hydrolase protein could be detected by western blotting. Molecular analysis in both patients uncovered mutations in the HIBCH gene, including one missense mutation in a conserved part of the protein and two mutations affecting splicing. A carefully interpreted acylcarnitine profile will allow more patients with 3-hydroxyisobutyryl-CoA hydrolase deficiency to be diagnosed.

Base Sequence↗

PubMed1999

Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase.

The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M. tuberculosis, but not in the vaccine strain Mycobacterium bovis BCG. It is a functional L-alanine dehydrogenase (EC 1.4.1.1) and hence one of the few antigens possessing an enzymic activity. This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions. Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme. In the oxidative-deamination reaction, the enzyme showed K(m) values of 13. 8 mM and 0.31 mM for L-alanine and NAD(+), respectively, in a random-ordered mechanism. K(m, app) values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 microM for ammonium, pyruvate and NADH, respectively. The enzyme is highly specific for all of its substrates in both directions. The pH profile indicates that oxidative deamination virtually may not occur at physiological pH. Hence L-alanine most likely is the product of the reaction catalysed in vivo. The enzyme is heat-stable, losing practically no activity at 60 degrees C for several hours.

Alanine Dehydrogenase↗

PubMed2005

Why do lipid transfer protein-hypersensitive patients tolerate bean (and other legumes)?

BACKGROUND: Patients allergic to nonspecific lipid transfer protein (LTP) frequently score positive on SPT with legumes but virtually never report adverse reactions eating these foods. OBJECTIVE: This study investigated the IgE reactivity to legumes of LTP-allergic patients and aimed to establish whether legumes can be considered safe in LTP-allergic patients. METHODS: Skin reactivity as well as clinical allergy to bean and pea were evaluated in a large cohort of LTP-hypersensitive patients. Sera from 12 patients showing clinical allergy to a number of botanically unrelated plant-derived foods and high levels of IgE to peach LTP were employed in in vitro studies (ELISA, ELISA inhibition, SDS-PAGE/immunoblot) aiming to investigate IgE reactivity to bean. RESULTS: Preabsorption of patients' sera with boiled bean extract did not cause any loss of IgE reactivity to peach LTP (whereas boiled apple totally abolished it). Immunoblot analysis did not show any IgE reactivity to bean proteins at about 10 kDa, and the SDS profile of bean showed little or no staining at 10 kDa. CONCLUSION: Sera from LTP-allergic patients do not seem to show any IgE reactivity to 10-kDa proteins in bean. Whether this is caused by epitopic differences between Rosaceae and bean LTPs or by the fact that LTP is not expressed in bean remains to be established. This study explains why virtually all LTP-allergic patients tolerate legumes and suggests that these foods should be considered safe for patients sensitized to this protein.

Antigens, Plant↗

PubMed2004

Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP kinase-dependent gene expression in TNF-alpha-stimulated primary human endothelial cells.

Inflammatory stimulation of endothelial cells by tumor necrosis factor alpha (TNF-alpha) involves activation of nuclear factor kappa B (NF-kappa B) and p38 mitogen-activated protein (MAP) kinase signaling pathways. A reliable analysis of the gene expression program elicited by TNF-alpha and its assignment to distinct signaling pathways is not available. A sophisticated analysis of oligonucleotide microarrays covering more than 13 000 genes allowed definition of the TNF-alpha-regulated endothelial gene expression profile and novel TNF-alpha-induced genes. Virtually all TNF-alpha-inducible genes were dependent on I kappa B kinase 2 (IKK2)/NF-kappa B activation, whereas a minor number was additionally modulated by p38. Furthermore, genes suppressed by IKK2/NF-kappa B were newly identified. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry confirmed reliability of data. Thus, these results define a list of primary candidates for targeted modulation of endothelial functions during inflammation.

Cells, Cultured↗