Disruption of ptLPD1 or ptLPD2, Genes That Encode Isoforms of the Plastidial Lipoamide Dehydrogenase,Confers Arsenate Hypersensitivity in Arabidopsis

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.Arsenic (As) is a naturally occurring metalloid found in soil, water, and air, but anthropogenic activities, including smelting and fossil fuel combustion, have led to increased environmental exposure (Mandal and Suzuki, 2002). In the environment, As exists in both organic and inorganic forms. Arsenate [As(V)] is the principal inorganic form of As in aerobic soils, while arsenite [As(III)] is the main form found under anaerobic conditions (Marin et al., 1993; Onken and Hossner, 1995, 1996; Mandal and Suzuki, 2002; Masscheleyn et al., 2002).Both As(V) and As(III) are toxic to plants, inducing symptoms ranging from poor seed germination and inhibited root growth to death (Meharg and Hartley-Whitaker, 2002; Lee et al., 2003; Ahsan et al., 2008; Smith et al., 2010). The modes of action of As(V) and As(III) differ, owing to their distinct chemical properties. As(V), with its structural similarity to phosphate, can compete with phosphate in oxidative phosphorylation, leading to the production of ADP-As(V) (Gresser, 1981). However, half-maximal stimulation of ADP-As(V) formation requires physiologically unlikely concentrations of approximately 0.8 mm As(V) (Moore et al., 1983). As(V) has been recently shown to enhance membrane fluidity, and thus membrane permeability, by binding and replacing phosphate or choline head groups (Tuan et al., 2008). The resulting damage to the membrane would disrupt the transport of mineral nutrients and water (Smith et al., 2010). As(V) can be promptly reduced in plants, including Arabidopsis (Arabidopsis thaliana), to As(III) by endogenous As(V) reductases, so that often more than 90% of As in plant cells is in the form of As(III) (Zhao et al., 2009). As(III) readily forms covalent bonds with sulfhydryl groups, especially vicinal dithiols. Binding to the free thiols of proteins is believed to be the basis of As(III) toxicity, either by inhibiting activity directly or by disrupting protein structure. Many enzymes have been proposed to be targets leading to As(III) toxicity, and the As(III) sensitivity of some of these enzymes has been investigated in nonplant systems (Adamson and Stevenson, 1981; Cavigelli et al., 1996; Lynn et al., 1997; Hu et al., 1998; Kitchin and Wallace, 2008). Of the many potential protein targets, only the pyruvate dehydrogenase complex (PDC) has been shown to be inactivated by physiologically relevant micromolar concentrations of As(III) (Hu et al., 1998), suggesting that PDC may be the primary target for As(III)-mediated cytotoxicity. However, little is known about the mechanism of As toxicity in vivo, especially in plants.Although As is phytotoxic, some plants species are resistant to high levels of As through avoidance mechanisms, while species of the Pteridaceae family of ferns hyperaccumulate As without toxic effects (Verbruggen et al., 2009; Zhao et al., 2009). As an analog of phosphate, As(V) is readily taken up by plants through high-affinity phosphate transporters encoded by the PHOSPHATE TRANSPORTER1 (PHT1) gene family (Shin et al., 2004; González et al., 2005; Catarecha et al., 2007). Except for the hyperaccumulating ferns, avoidance of As toxicity by resistant species is often accomplished by a decrease in phosphate uptake activity (Meharg and Hartley-Whitaker, 2002). Unlike As(V), the transport of As(III) is facilitated by aquaporin nodulin 26-like intrinsic proteins (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008; Kamiya et al., 2009). In roots and fronds of hyperaccumulating ferns, As(III) is sequestered in the vacuole (Lombi et al., 2002; Pickering et al., 2006). Much of the As(III) taken up by nonaccumulating resistant species may be released back to the rhizosphere through an undefined efflux pathway (Zhao et al., 2009). As(III) that remains in tissues reacts with thiol-containing molecules, such as glutathione or phytochelatins, both of which are usually produced in greater abundance in response to As (Grill et al., 1987; Sneller et al., 1999; Schmöger et al., 2000; Schulz et al., 2008). As(III)-glutathione adducts can be sequestered in the vacuole (Dhankher et al., 2002; Bleeker et al., 2006). However, increased synthesis of glutathione or phytochelatins alone is unlikely to confer a very high level of tolerance (Zhao et al., 2009).To identify genes essential for As resistance in plants, we used a genetic screen to identify mutants of Arabidopsis that were hypersensitive to As(V). The screen was analogous to that used to isolate the salt overly sensitive (sos) mutants of Arabidopsis (Wu et al., 1996) that led to the identification of the SOS pathway for salt tolerance (Zhu, 2000, 2003). Our hypothesis was that arsenate overly sensitive (aos) mutants would reveal a different set of genes from those identified in mutants showing increased resistance to As(V).     

Chloroplast DNA inheritance and variation in <Emphasis Type="Italic">Leucadendron</Emphasis> species (Proteaceae) as revealed by PCR-RFLP

The inheritance of chloroplast DNA (cpDNA) in Leucadendron species was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. A total of 100 progeny from five interspecific crosses involving seven parental species were tested, and all progeny exhibited the cpDNA restriction fragment pattern of the female parent, indicating that cpDNA in Leucadendron is maternally inherited. PCR-RFLP was also employed to study cpDNA variation among 21 Leucadendron species. Parsimony analysis using a heuristic search resulted in a phylogenetic tree that showed limited agreement to the taxonomic classification of Leucadendron species, based on morphological characteristics. The incongruence between cpDNA phylogenetic and taxonomic groupings in Leucadendron may be due to reticulate evolution involving a combination of hybridization and introgression, convergent evolution and/or lineage sorting at the interspecific, intersubsectional and intersectional levels.     

Expression of CD80/86 on B cells is essential for autoreactive T cell activation and the development of arthritis

Depletion of B cells in rheumatoid arthritis is therapeutically efficacious. Yet, the mechanism by which B cells participate in the inflammatory process is unclear. We previously demonstrated that Ag-specific B cells have two important functions in the development of arthritis in a murine model of rheumatoid arthritis, proteoglycan (PG)-induced arthritis (PGIA). PG-specific B cells function as autoantibody-producing cells and as APCs that activate PG-specific T cells. Moreover, the costimulatory molecule CD86 is up-regulated on PG-specific B cells in response to stimulation with PG. To address the requirement for CD80/CD86 expression on B cells in the development of PGIA, we generated mixed bone marrow chimeras in which CD80/CD86 is specifically deleted on B cells and not on other APC populations. Chimeras with a specific deficiency in CD80/CD86 expression on B cells are resistant to the induction of PGIA. The concentration of PG-specific autoantibody is similar in mice sufficient or deficient for CD80/86-expressing B cells, which indicates that resistance to PGIA is not due to the suppression of PG-specific autoantibody production. CD80/86-deficient B cells failed to effectively activate PG-specific autoreactive T cells as indicated by the failure of T cells from PG-immunized CD80/86-deficient B cell chimeras to transfer arthritis into SCID mice. In vitro secondary recall responses to PG are also dependent on CD80/86-expressing B cells. These results demonstrate that a CD80/86:CD28 costimulatory interaction between B cells and T cells is required for autoreactive T cell activation and the induction of arthritis but not for B cell autoantibody production.     

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

Self-incompatibility: Smi silences through a novel sRNA pathway

Self-incompatibility in Brassicaceae is determined by the interaction between S-Locus Protein 11 (SP11) on the pollen and S-receptor kinase (SRK) in the stigma. Pollen from heterozygotes generally displays products of both SP11 alleles, but in some heterozygotes SP11 expression is monoallelic, with one allele (SP11(R)) being silenced by promoter methylation. An exciting development in understanding the mechanism behind monoallelic silencing came recently when Y. Tarutani et al. [Nature 2010;466:983-986] identified a 24-nucleotide sRNA (termed Smi) derived from a non-coding gene within the dominant S-haplotype, and suggested that Smi directs promoter methylation. We propose that rather than having a direct effect on DNA methylation, Smi is the first step in a novel cis-acting siRNA pathway that directs widespread monoallelic SP11(R) promoter methylation.     

Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.     

T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system

T cell homeostasis is a physiological function of the immune system that maintains a balance in the numbers and ratios of T cells at the periphery. A self-MHC/self-peptide ligand can induce weak (covert) signals via the TCR, thus providing an extended lifespan for naive T cells. A similar mechanism is responsible for the restoration of immune homeostasis in severe lymphopenic conditions such as those following irradiation or chemotherapy, or upon transfer of lymphocytes to nu/nu or SCID mice. To date, the genetic backgrounds of donor and recipient SCID mice were unmatched in all autoimmune arthritis transfer experiments, and the recovery of lymphoid cells in the host has not been followed. In this study, we present the adoptive transfer of proteoglycan (PG)-induced arthritis using unseparated and T or B cell-depleted lymphocytes from arthritic BALB/c donors to genetically matched syngeneic SCID recipient mice. We demonstrate that selectively recovered lymphoid subsets determine the clinical and immunological status of the recipient. We found that when T cells were depleted (>98% depleted), B cells did not produce PG-specific anti-mouse (auto) Abs unless SCID mice received a second Ag (PG) injection, which promoted the recovery of Ag-specific CD4(+) Th1 cells. Reciprocally, as a result of B cell recovery, high levels of serum anti-PG Abs were found in SCID mice that received B cell-depleted (>99% depleted) T lymphocytes. Our results indicate a selective and highly effective cooperation between CD4(+) T cells and B lymphocytes that is required for the restoration of pathological homeostasis and development of autoimmune arthritis in SCID mice.