Donor T cell activation initiates small bowel allograft rejection through an IFN-gamma-inducible protein-10-dependent mechanism

The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2L(d+)) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (L(d)-deficient) BALB/c H-2(dm2) (dm2, H-2L(d-)) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA(323-339) peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas <20% of allografts were rejected in mice treated with control peptide (p < 0.05). Isografts survived >30 days regardless of OVA(323-339) administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-gamma) and CXC chemokine IFN-gamma-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-gamma-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.     

Hydrogen peroxide and nitric oxide as signalling molecules in plants

It is now clear that hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) function as signalling molecules in plants. A wide range of abiotic and biotic stresses results in H(2)O(2) generation, from a variety of sources. H(2)O(2) is removed from cells via a number of antioxidant mechanisms, both enzymatic and non-enzymatic. Both biotic and abiotic stresses can induce NO synthesis, but the biosynthetic origins of NO in plants have not yet been resolved. Cellular responses to H(2)O(2) and NO are complex, with considerable cross-talk between responses to several stimuli. In this review the potential roles of H(2)O(2) and NO during various stresses and the signalling pathways they activate are discussed. Key signalling components that might provide targets for enhancing crop production are also identified.     

Which way to manipulate host reproduction? Wolbachia that cause cytoplasmic incompatibility are easily invaded by sex ratio-distorting mutants

The bacterium Wolbachia manipulates its hosts by inducing cytoplasmic incompatibility (CI), where zygotes formed from crosses between uninfected mothers and infected fathers die. In addition, it distorts the host's sex ratio via male killing, parthenogenesis induction, or feminization. Here, we model transitions between these states, examining the evolution of mutants of CI strains that retain both the ability to induce and resist CI but, in addition, cause sex ratio distortion. The model shows that CI strains are highly susceptible to invasion and subsequent elimination by these mutants. For all three types of sex ratio distortion, there is some parameter space in which the strain showing sex ratio distortion becomes extinct following exclusion of the progenitor CI strain, leaving the population uninfected. Extinction of the new Wolbachia strain is common for the case of male killing but rarer for parthenogenesis induction and feminization. Our models predict that CI strains of Wolbachia will occur most commonly in hosts that are male heterogametic, where there is little interaction between siblings because these hosts are unlikely to favor the spread of male killing, feminization, or parthenogenesis induction. The models raise the question of why CI strains apparently predominate in nature, and it is suggested that this is a result of either fewer restrictions on CI strains spreading through novel host populations or restrictions to the mutability of Wolbachia strains.     

Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens

Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, or sepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.     

Permeation of phospholipid membranes by peroxynitrite

Quantitative kinetic models have been developed for the reaction between peroxynitrite and membrane lipids in vesicles and for transmembrane oxidation of reactants located within their inner aqueous cores. The models were used to analyze TBARS formation and oxidation of entrapped Fe(CN)(6)(4)(-) ion in egg lecithin liposomes and several artificial vesicles. The analyses indicate that permeation of the bilayers by ONOOH and NO(2)(*), a radical formed by homolysis of the ONOOH bond, is unusually rapid but that permeation by ONOO(-) and CO(3)(*)(-), a radical formed when CO(2) is present, is negligible. Bicarbonate protects the vesicles against both membrane and Fe(CN)(6)(4)(-) oxidation by rapid competitive CO(2)-catalyzed isomerization of ONOOH to NO(3)(-); this effect is partially reversed by addition of nitrite ion, which reacts with CO(3)(*)(-) to generate additional NO(2)(*). Under medium conditions mimicking the physiological milieu, a significant fraction of the oxidants escape to inflict damage upon the vesicular assemblies. Rate constants for several elementary reaction steps, including transmembrane diffusion rates for ONOOH and NO(2)(*), were estimated from the bicarbonate dependence of the oxidative reactions.     

Development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues

Protegrin antimicrobial peptides possess activity against gram-positive and gram-negative bacteria and yeasts. An extensive structure-activity relationship (SAR) study was conducted on several hundred protegrin analogues to gain understanding of the relationship between the primary and secondary structure of the protegrins and their antimicrobial activities, and to identify a protegrin analogue for clinical development. Native sequence protegrins are cationic, amphiphilic peptides that are characterized by the presence of a beta-sheet structure that is maintained by two disulfide bridges. The presence of the beta-sheet is key to the stability of the protegrin structure; linearized analogues or analogues that have amino acid substitutions that eliminate hydrogen bonding across the beta-sheet have reduced activity, especially in the presence of physiological concentrations of NaCl. Also, maintaining amphiphilicity of the beta-sheet is key; analogues with substitutions of polar amino acids in the hydrophobic face have reduced activity. Analogues with reduced positive charge tend to be less active, an observation that is more marked for gram-negative than gram-positive bacteria, and may implicate binding to lipopolysaccharide as a key mechanistic step in the killing of gram-negative bacteria. A very large number of amino acid substitutions are tolerated by the protegrin structure, implying that overall structural features such as amphiphilicity, charge, and shape are more important to activity than the presence of specific amino acids. This lack of importance of specific stereochemistry is supported by the fact that completely D-amino acid substituted protegrins are fully potent. Based on the SAR studies, and on the microbiological data from an animal model, one protegrin analogue, IB-367, was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy.     

NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Molecular evolution of an imprinted gene: repeatability of patterns of evolution within the mammalian insulin-like growth factor type II receptor.

The repeatability of patterns of variation in Ka/Ks and Ks is expected if such patterns are the result of deterministic forces. We have contrasted the molecular evolution of the mammalian insulin-like growth factor type II receptor (Igf2r) in the mouse-rat comparison with that in the human-cow comparison. In so doing, we investigate explanations for both the evolution of genomic imprinting and for Ks variation (and hence putatively for mutation rate evolution). Previous analysis of Igf2r, in the mouse-rat comparison, found Ka/Ks patterns that were suggested to be contrary to those expected under the conflict theory of imprinting. We find that Ka/Ks variation is repeatable and hence confirm these patterns. However, we also find that the molecular evolution of Igf2r signal sequences suggests that positive selection, and hence conflict, may be affecting this region. The variation in Ks across Igf2r is also repeatable. To the best of our knowledge this is the first demonstration of such repeatability. We consider three explanations for the variation in Ks across the gene: (1) that it is the result of mutational biases, (2) that it is the result of selection on the mutation rate, and (3) that it is the product of selection on codon usage. Explanations 2 and 3 predict a Ka-Ks correlation, which is not found. Explanation 3 also predicts a negative correlation between codon bias and Ks, which is also not found. However, in support of explanation 1 we do find that in rodents the rate of silent C --> T mutations at CpG sites does covary with Ks, suggesting that methylation-induced mutational patterns can explain some of the variation in Ks. We find evidence to suggest that this CpG effect is due to both variation in CpG density, and to variation in the frequency with which CpGs mutate. Interestingly, however, a GC4 analysis shows no covariance with Ks, suggesting that to eliminate methyl-associated effects CpG rates themselves must be analyzed. These results suggest that, in contrast to previous studies of intragenic variation, Ks patterns are not simply caused by the same forces responsible for Ka/Ks correlations.