IL-2R beta agonist P1-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological and molecular effects

From the sequence of human IL-2 we have recently characterized a peptide (p1-30), which is the first IL-2 mimetic described. P1-30 covers the entire alpha helix A of IL-2 and spontaneously folds into a alpha helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor beta-chain likely to form the p1-30 receptor (p1-30R). P1-30 acts as a specific IL-2Rbeta agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1-30 induces the activation of lymphokine-activated killer cells and the production of IFN-gamma. Here we demonstrate the ability of p1-30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1beta, a murine T cell line endogenously expressing the common cytokine gamma gene and transfected with the human IL-2Rbeta gene. At the receptor level, we show that expression of human IL-2Rbeta is absolutely required to obtain synergistic effects, whereas IL-2Ralpha specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Ralpha inhibits p1-30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1-30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1-30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1-30R (IL-2Rbeta)(2) and intermediate affinity IL-2R (IL-2Rbetagamma), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.     

Synthesis of glyoxylyl peptides using an Fmoc-protected alpha,alpha'-diaminoacetic acid derivative.

The synthesis of glyoxylyl peptides by coupling the masked glyoxylic acid derivative (FmocNH)(2)CHCO(2)H, 1, to a peptidyl resin assembled using Fmoc/tert-butyl chemistry has been described recently. Deprotection and cleavage of the peptide from the solid support using TFA was followed by unmasking of the glyoxylyl group in solution in the presence of DBU. [] The glyoxylyl peptide was thus generated using non-oxidizing conditions by comparison with the method based on the periodic oxidation of a seryl-precursor. However, base treatment of the (FmocNH)(2)CHCO(2)-peptide led to the formation of a byproduct besides the desired glyoxylyl peptide. This paper describes an optimized procedure for unmasking the Fmoc-protected alpha,alpha'-diaminoacetic acid moiety in solution which suppressed byproduct formation. Also presented is a series of experiments that permitted a structure and a mechanism of formation for the byproduct to be suggested.     

Ethical,legal and social aspects of the approach in Sudan

The global malaria situation, especially in Africa, and the problems frequently encountered in chemical control of vectors such as insecticide resistance, emphasize the urgency of research, development and implementation of new vector control technologies that are applicable at regional and local levels. The successful application of the sterile insect technique (SIT) for the control of the New World screwworm Cochliomyia hominivorax and several species of fruit flies has given impetus to the use of this method for suppression or elimination of malaria vectors in some areas of Africa including Northern State of Sudan. The research and development phase of the Northern State feasibility study has been started. Sudanese stakeholders are working side-by-side with the International Atomic Energy Agency in the activities of this important phase. Several ethical, legal and social issues associated with this approach arose during this phase of the project. They need to be seriously considered and handled with care. In this paper, these issues are described, and the current and proposed activities to overcome potential hurdles to ensure success of the project are listed.     

Complete Genome Sequence of the Electricity-Producing “Thermincola potens” Strain JR

“Thermincola potens” strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR.“Thermincola potens” strain JR, a Gram-positive anaerobe isolated from a thermophilic microbial fuel cell (MFC), constituted a dominant member of the current-producing bacterial community (10). Strain JR is a Thermincola species in the phylum Firmicutes belonging to the family Peptococcaceae in the order Clostridiales. It shares 99% 16S rRNA gene sequence identity with the two known members of the Thermincola genus, T. carboxdophilia and T. ferriacetica (8, 12). This strain coupled acetate oxidation to reduction of the insoluble electron acceptors MFC anodes and hydrous ferric oxide (HFO) (10). Strain JR is also capable of growth with CO as the sole electron donor and carbon source.This member of the Firmicutes is the first MFC isolate and Thermincola species to have its genome sequenced and is one of only a few bacteria in the Peptococcaceae to have its genome sequenced (5, 11). Genomic analysis will aid elucidation of electron transfer mechanisms by strain JR, contributing to the knowledge of extracellular respiration by Gram-positive bacteria. By comparing these mechanisms to those in Gram-negative organisms, the conserved and disparate aspects of this seminal metabolism can be identified. This will include analysis of the c-type cytochrome gene makeup of the genome, especially the increased number of proteins with double heme (CXXCH) motifs and multiple heme binding domains compared to the nearest phylogenetic neighbors with sequenced genomes (4, 6, 7). c-type cytochromes are essential for the reduction of insoluble electron acceptors by model Gram-negative bacteria, such as Geobacter or Shewanella species (3, 9); however, their role in Gram-positive mineral respiration is still unknown.Joint Genome Institute (JGI) sequencing used a combination of 454 and Illumina techniques with 27× coverage. All library construction and sequencing techniques are available at http://www.jgi.doe.gov/. Illumina reads were assembled into 121 contigs using Velvet 0.7.1.18 (13) and shredded into 1-kb pseudoreads (with 100-bp overlap). The pseudoreads were incorporated into a hybrid 454/Illumina assembly using the parallel Phrap assembler (CodonCode Corporation, Dedham, MA) (1, 2). Misassemblies were corrected with Dupfinisher (C. S. Han and P. Chain, presented at the 2006 International Conference on Bioinformatics and Computational Biology). Gene modeling was performed using Prodigal (http://prodigal.ornl.gov/), and resulting protein translations were assigned by comparisons to Pfam, KEGG, and COGs databases using BLASTP or HMMER. The complete genome was a single circular chromosome of approximately 3,036,819 bp with an average G+C content of 45.9%. A total of 2,963 protein-encoding genes were predicted, and 393 (6.9%) had no similarity to public database sequences.     

Adaptive landscapes in evolving populations of Pseudomonas fluorescens

The repeatability of adaptive evolution depends on the ruggedness of the underlying adaptive landscape. We contrasted the relative ruggedness of two adaptive landscapes by measuring the variance in fitness and metabolic phenotype within and among genetically distinct strains of Pseudomonas fluorescens in two environments differing only in the carbon source provided (glucose vs. xylose). Fitness increased in all lines, plateauing in one environment but not the other. The pattern of variance in fitness among replicate lines was unique to the selection environment; it increased over the course of the experiment in xylose but not in glucose. Metabolic phenotypes displayed two results: (1) populations adapted via changes that were distinctive to their selection environment, and (2) endpoint phenotypes were less variable in glucose than in xylose. These results indicate that although the response to selection is highly repeatable at the level of fitness, the underlying genetic routes taken were different for each environment and more variable in xylose. We suggest that this reflects a more rugged adaptive landscape in xylose compared to glucose. Our study demonstrates the utility of using replicate selection lines with different evolutionary starting points to try and quantify the relative ruggedness of adaptive landscapes.     

Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.