An alternative mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.     

IL-15 and IL-2: a matter of life and death for T cells in vivo

Interleukin (IL)-2 and IL-15 are redundant in stimulating T-cell proliferation in vitro. Their precise role in vivo in governing T-cell expansion and T-cell homeostasis is less clear. Each may have distinct functions and regulate distinct aspects of T-cell activation. The functional receptors for IL-2 and IL-15 consist of a private alpha-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor beta- and gamma-chains. The gamma-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the gamma-chain is called the common gamma or gamma-c. As these receptor subunits can be expressed individually or in various combinations resulting in the formation of receptors with different affinities, distinct signaling capabilities or both, we hypothesized that differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, we demonstrate that IL-15 is a critical growth factor in initiating T cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via downregulation of the gamma-c expression. Decreased gamma-c expression on cycling T cells reduced sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Our study provides data that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo.     

Characterisation of set-1, a conserved PR/SET domain gene in Caenorhabditis elegans

We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.     

Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.     

Competition for Astragalus sinicus root nodule infection between its native microsymbiont Rhizobium huakuii bv. renge B3 and Rhizobium sp. ACMP18 strain, specific for Astragalus cicer

Rhizobium huakuii bv. renge B3, a native symbiont of Astragalus sinicus, outcompeted Rhizobium sp. strain ACMP18, which was isolated from Astragalus cicer nodules, in the formation of root nodules on A.␣sinicus when plants were co-inoculated with these strains. The strains occupying the nodules were identified by antibiotic resistance and phage sensitivity markers and also by polymerase chain reaction (PCR) genomic fingerprintings, which were performed by using enterobacterial repetitive intergenic consensus sequences. In PCR genomic fingerprintings, the total genomic DNA isolated from pure bacterial culture and from squashed root nodules showed identical profiles, indicating that this technique can be a useful tool for identification of rhizobia in ecological studies. When Rhizobium sp. strain ACMP18 outnumbered R. huakuii bv. renge strain B3 by a factor of ten, and even when strain ACMP18 was added to plants 1 week before bacterization with strain B3, the strain B3 occupied most nodules. Dually infected nodules were not observed, although Rhizobium sp. ACMP18 formed active nodules on A. sinicus when the bacterial strain was inoculated alone. Received: 5 February 1998 / Received revision: 23 March 1998 / Accepted: 27 April 1998