The EBNA-2 arginine-glycine domain is critical but not essential for B-lymphocyte growth transformation; the rest of region 3 lacks essential interactive domains.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.     

Ciprofibrate therapy in patients with hypertriglyceridemia and low high density lipoprotein (HDL)-cholesterol: greater reduction of non-HDL cholesterol in subjects with excess body weight (The CIPROAMLAT study)

Background

Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk.

Methods

The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion.

Results

In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246).

Conclusion

The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.     

Stabilization of the soluble,cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schülke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.     

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif

Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.     

Changes in extracellular pH are neither required nor sufficient for activation of mitogen-activated protein kinases (MAPKs) in response to systemin and fusicoccin in tomato

Leaf wounding and the wound signaling peptide systemin induce expression of wound response genes while the fungal toxin fusicoccin (FC) induces expression of pathogenesis-related genes. Consistent with their functional differences, FC and systemin regulate the extracellular pH in opposite ways, with systemin inducing an alkalinization and FC an acidification response. Here we show that systemin, wounding and FC activate the same mitogen-activated protein kinases (MAPKs; MPKs) MPK1 and 2 in tomato (Lycopersicon esculentum) leaves and L. peruvianum suspension-cultured cells. Wounding and FC activated an additional MAPK, MPK3. Pronounced differences were observed with regard to MAPK activation kinetics. FC induced prolonged, and systemin transient activity of the MAPKs. This shows that functionally different elicitors engage the same signaling components, yet induce signal-specific activation dynamics. A comparative analysis of pH effects and MAPK activity in response to specific treatments revealed that the kinetics of pH changes and MAPK activation did not correlate. Simultaneous application of FC and systemin did not lead to immediate pH changes but resulted in rapid increases in MAPK activity. Furthermore, changes in extracellular pH could be induced without concomitant MAPK activation by exchanging conditioned medium with fresh medium. This shows that changes in the extracellular pH are neither required nor sufficient for MAPK activation, suggesting that signaling pathways involving MAPKs and extracellular pH changes operate in parallel and are not part of the same linear pathway.     

Radiolabeled divalent peptidomimetic vitronectin receptor antagonists as potential tumor radiotherapeutic and imaging agents

The integrin receptor alphavbeta3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled alphavbeta3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin alphavbeta3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 A) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.