Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear.

ProtoClear is a proprietary technique for clearing albumin and immunoglobulin G (IgG) from human serum samples. Albumin constitutes 57-71% of total serum protein and IgG ranges from 8-26%. Removal of these two proteins alone clears approximately 75% of the total protein present in serum and allows the detection of the remaining proteins that are present in far lower concentrations. ProtoClear effectively removed >95% of human serum albumin (HSA) and >97% of human IgG as measured by an anti-HSA competitive immunoassay and a radial immunodiffusion assay, respectively. ProtoClear was far more specific at removing albumin and IgG than Cibracon Blue Dye chromatography (Cibracon Blue), the typically utilized alternative. Comparing two-dimensional (2-D) gels of serum cleared by either Cibracon Blue or by ProtoClear, it was apparent that Cibracon Blue removed a number of proteins in addition to albumin. Following removal of albumin and IgG from serum, we found a significant improvement in the resolution of polypeptide spots detected on two-dimensional gels.

Inhibition of synoviocyte collagenase gene expression by adenosine receptor stimulation.

OBJECTIVE: To characterize the regulation of matrix metalloproteinases (MMPs) by adenosine. METHODS: Cultured fibroblast-like synoviocytes (FLS) were stimulated with interleukin-1 (IL-1) in the presence or absence of adenosine receptor agonists. Immunoreactive MMPs were measured using specific enzyme-linked immunosorbent assays, and gene expression was assessed by Northern blot analysis. RESULTS: The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) decreased collagenase production by IL-1-stimulated synoviocytes from 196 +/- 28 ng/ml (mean +/- SEM) to 66 +/- 9 ng/ml (P < 0.001). There was minimal effect on stromelysin production (decrease from 107 +/- 16 ng/ml to 97 +/- 15 ng/ml). Selective adenosine receptor agonists implicated the A2b adenosine receptor in this activity, and reverse transcriptase-polymerase chain reaction studies confirmed that FLS express this receptor. Northern blot analysis demonstrated that the mechanism of action was pre-translational since NECA decreased collagenase, but not stromelysin or tissue inhibitor of metalloproteinases 1 (TIMP-1), messenger RNA levels. Cyclic AMP levels were increased by NECA, and a direct adenylate cyclase activator (forskolin) also suppressed collagenase gene expression. These data suggest that cAMP mediates the inhibitory effect of NECA on collagenase production. CONCLUSION: Stimulation of the A2b receptor on FLS decreases collagenase gene expression, with little or no effect on stromelysin and TIMP-1. The combination of antiinflammatory and MMP-regulating properties of adenosine or adenosine-regulating agents suggest that treatment based on this approach might be useful in rheumatoid arthritis.

Inhibition of TNF-alpha expression by adenosine: role of A3 adenosine receptors.

Adenosine agonists inhibit TNF-alpha production in macrophage and monocytes, but the mechanism is unknown. Therefore, we studied the human macrophage cell line U937 to determine the adenosine receptor subtypes responsible and the intracellular signaling mechanisms involved. The A1/A3 agonist N6-(4-amino-3-iodobenzyl)adenosine (I-ABA) decreased LPS-stimulated TNF-alpha protein production by 79 +/- 5% (p = 0.003). The mechanism was pretranslational, as adenosine receptor stimulation caused a marked decrease in TNF-alpha mRNA. IL-1 beta, IL-6, and IL-8 mRNA were not changed by adenosine agonists. The rank order of agonists as TNF-alpha inhibitors suggested that the A3 receptor might be involved (N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-beta-D-ribofuranosyl] adenosine > 2-chloroadenosine > or = I-ABA > N6 benzyl 5'-N-ethylcarboxamidoadenosine (NECA) > NECA > CGS21680 > N6-cyclohexyladenosine), and this was supported by the fact that a mixed A1/A3 antagonist (xanthine amine congener) reversed the effect, whereas A1-specific (1,3-dipropyl-8-cyclopentylxanthine) and A2-specific (3,7-dimethyl-1-propargylxanthine) antagonists did not. Receptor signaling did not involve cAMP or protein kinase A, nor did it alter the activation and binding characteristics of the transcription factor NF-kappa B. However, the composition of the AP-1 transcription complex was altered by I-ABA. These data suggest that stimulation of the A3 adenosine receptor can alter the cytokine milieu by decreasing TNF-alpha. Adenosine agonists or adenosine regulating agents have potential therapeutic uses in acute and chronic inflammatory diseases.

cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant.

A cDNA encoding variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full-length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gi proteins. Analysis of receptor transcripts by RT-PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors.

Protective effect of an adenosine kinase inhibitor in septic shock.

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.

cDNA cloning and sequence analysis of the human A3 adenosine receptor.

We report the cloning of the cDNA encoding the homolog of the A3 adenosine receptor (A3AR) from a human heart library. The receptor shares a surprisingly low degree of sequence homology to the rat A3AR. Northern analysis of various human tissues showed the human A3AR gene to be expressed primarily in lung, liver, kidney and heart, with very little expression in the brain or in skeletal muscle.

Five novel avian Eph-related tyrosine kinases are differentially expressed.

We have identified cDNA clones that encode five new avian receptor-like tyrosine kinases of the Eph subclass, by screening two chicken embryonic cDNA libraries with DNA probes. We have designated them Cek6 to Cek10. The identification of these kinases indicates that the Eph subclass comprises at least 10 members and, therefore, represents a very large family of receptor-like tyrosine kinases. Variants of Cek10 and of Cek5 (a previously identified Eph-related kinase) containing amino acid insertion sequences in the juxtamembrane domain were also isolated. The Cek5 variant is expressed in the brain, but not in other tissues of the 10-day chick embryo. Analysis of 10-day chick embryo mRNAs shows the newly identified tyrosine kinases to be all expressed in both the embryonic brain and body tissues. In adult tissues, they display distinct patterns of expression. Cek6, Cek7, Cek8, Cek9 and Cek10 are likely to play significant roles in embryonic signal transduction pathways, including those involved in neural development. Their distinct tissue distributions in the adult suggest that the different members of the Eph family may each serve specific functions.

Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.

Ciprofibrate, a hypolipidemic drug that acts as a peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs of promoter sequence from the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream of a luciferase reporter gene. Transfection of this expression vector into rat hepatoma H4IIEC3 cells in the presence of ciprofibrate resulted in a 5- to 10-fold, cell type-specific increase in luciferase activity as compared to cells transfected in the absence of drug. A peroxisome proliferator-responsive element (PPRE) was localized to a 196-nucleotide region centered at position -2943 from the transcription start site. This PPRE conferred ciprofibrate responsiveness on a heterologous promoter and functioned independently of orientation or position. Gel retardation analysis with nuclear extracts demonstrated that ciprofibrate-treated or untreated H4IIEC3 cells, but not HeLa cells or monkey kidney cells, contained sequence-specific DNA binding factors that interact with the PPRE. These results have implications for understanding the mechanisms of coordinated transcriptional induction of genes encoding peroxisomal proteins by hypolipidemic agents and other peroxisome proliferators.

Identification of a new eph-related receptor tyrosine kinase gene from mouse and chicken that is developmentally regulated and encodes at least two forms of the receptor.

We have identified cDNA clones that encode a new member of the Eph receptor tyrosine kinase subclass by using antibodies to phosphotyrosine to screen a chicken embryonic cDNA expression library. These cDNAs were in turn used to isolate the entire coding sequence of this receptor from both mouse and chicken (designated Mek4 and Cek4, respectively). A cDNA that encodes a putative secreted form of the murine receptor's ligand-binding domain and lacks the transmembrane and kinase domains was also isolated. Analysis of the appropriate Mek4 genomic region revealed the presence of sequences required for the production of the secreted form of the receptor. Analysis of RNAs from various tissues shows the receptor to be highly expressed in mouse and chicken embryos, with the greatest levels of expression occurring in the brain. In adult mouse, the pattern of RNA expression is altered and appears to be confined primarily to the brain. However, a shorter transcript was found to be expressed at reduced levels in the testis. This new Eph-related receptor may play an important role during development and in signal transduction pathways.

Analysis of origin of DNA replication of human papovavirus BK.

We have analyzed the cis-acting sequence elements and properties of the origin of DNA replication of human papovavirus BK (BKV). The precise boundaries of the origin varied, depending on the cell type and the viral T antigen used for assay. The BKV minimal origin of replication consisted of an inverted repeat, T-antigen-binding site II, and a 20-base-pair AT block when assayed in monkey kidney CV1 and HeLa cells by using the BKV T antigen. This 76-base-pair minimal origin did not replicate in COS cells in the presence of the simian virus 40 (SV40) T antigen. Unlike that from the SV40 minimal origin, replication from the BKV minimal origin was not enhanced by BKV ori-flanking sequences in CV1 or HeLa cells, using the BKV T antigen. BKV ori-flanking sequences did activate the SV40 minimal origin of replication in COS cells and relieved the orientation-dependent property of this origin. Finally, the BKV T antigen was found to autoregulate activity of the BKV early transcriptional regulatory region. The BKV origin of replication shows similarities to and differences from those of the related viruses SV40 and polyomavirus, suggesting that the proteins involved in the initiation of replication interact with origin sequences differently in these viruses.

The modulatory element TNNCT affects transcription of a Drosophila tRNA(Val)4 gene without affecting transcription complex stability.

We have previously identified a sequence of the general form TNNCT in the 5'-flanking region of a Drosophila melanogaster tRNA(Val)4 gene which positively modulates in vitro transcription. The pentadeoxynucleotide is also present in the 5'-flank of other Drosophila tRNA genes which direct transcription efficiently in vitro. We have examined transcription of a mutant of this gene further to understand the role of the TNNCT sequence. When template competition experiments were carried out, a template which contained mutations in TNNCT was a better competitor than was the wildtype template. The time-course of transcription and the effects of temperature and ionic strength on transcription indicated that mutating the TNNCT did not alter the efficiency of stable complex formation. It is proposed that the pentadeoxynucleotide TNNCT may influence the rate of transcription initiation by RNA polymerase III bound to transcription complexes.

Modulation of a Drosophila melanogaster tRNA gene transcription in vitro by a sequence TNNCT in its 5' flank.

We have previously demonstrated the existence of both positive and negative transcription modulatory sequences in the 5' flank of a Drosophila melanogaster tRNA(4Val) gene using deletion analysis. The deletion of a pentadeoxynucleotide TCGCT between nucleotides (nt) -34 and -38 relative to the mature coding sequences resulted in a 90% decrease in template activity. In the present study, we have created a number of site-specific changes in the sequence TCGCT. Results indicate a significant decrease in the level of template activity when nt -38(T), -35(C) and -34(T) were mutated. In contrast, a change at nt -37(C) resulted in a slight increase in transcription and a change at nt -36(G) reduced template activity by only 1%. Sequences flanking other tRNA genes which are efficient templates for transcription were examined and found to contain a sequence closely related to TCGCT. We propose that a general form of the sequence TNNCT is a positive transcription modulator for a class of Drosophila tRNA genes.