IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

On the interaction of colicin E3 with the ribosome

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone     

Prediction of the structure of the complex between the 30S ribosomal subunit and colicin E3 via weighted-geometric docking

Colicin E3 kills Escherichia coli cells by ribonucleolytic cleavage in the 16S rRNA. The cleavage occurs at the ribosomal decoding A-site between nucleotides A1493 and G1494. The breaking of this single phosphodiester bond results in a complete termination of protein biosynthesis leading to cell death. A model structure of the complex of the ribosomal subunit 30S and colicin E3 was constructed by means of a new weighted-geometric docking algorithm, in which interactions involving specified parts of the molecular surface can be up-weighted, allowing incorporation of experimental data in the docking search. Our model, together with available experimental data, predicts the role of the catalytic residues of colicin E3. In addition, it suggests that bound acidic immunity protein inhibits the enzymatic activity of colicin E3 by electrostatic repulsion of the negatively charged substrate.     

Phosphohexose isomerase/autocrine motility factor/neuroleukin/maturation factor is a multifunctional phosphoprotein

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.     

The function and regulation of vasa-like genes in germ-cell development

The vasa gene, essential for germ-cell development, was originally identified in Drosophila, and has since been found in other invertebrates and vertebrates. Analysis of these vasa homologs has revealed a highly conserved role for Vasa protein among different organisms, as well as some important differences in its regulation.     

Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B

Glycerol triacetate was successfully used as a green solvent and as the acyl donor in the transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized Candida antarctica lipase B. Immobilized lipase was more catalytically active than free lipase and could be easily separated from the reaction mixture by filtration. In addition, it was found that increasing either the reaction temperature or the enzyme to substrate ratio increased the conversion of isoamyl alcohol. Using triacetin as the solvent also enabled the separation of product by simple extraction with petroleum ether and catalyst recycling.     

Galectin-3 in apoptosis,a novel therapeutic target

During the past decade, extensive progress has been made toward understanding the molecular basis for the regulation of apoptosis. In mammalian cells undergoing apoptosis, two distinct mechanisms or pathways are operated and are triggered by cell death stimuli from intra- or extra-cellular environments, namely the intrinsic or extrinsic pathways, resulting in mitochondrial membrane depolarization. Several lines of evidence from our laboratories and others have indicated that galectin-3 plays an important role in these pathways by binding to various ligands. Here the authors provide a brief discussion on the role of endogenous or extra-cellular galectin-3 in the regulation of apoptosis and how it could be used as a therapeutic target using natural plant products as its functional inhibitors.     

Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor

The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.