Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.     

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1alpha and IL-1beta), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1alpha (pIL-1alpha), mature IL-1beta (mIL-1beta), and mIL-1beta fused to a signal sequence (ssIL-1beta), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1alpha, which expresses IL-1alpha on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-gamma production. Cells transfected with secretable IL-1beta (mIL-1beta and ssIL-1beta) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1beta transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1beta tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1beta transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1beta transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-gamma and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1alpha tumor sites, whereas in mIL-1beta and ssIL-1beta tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1alpha and IL-1beta in malignant processes; IL-1alpha reduces tumorigenicity by inducing antitumor immunity, whereas IL-1beta promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.     

Accuracy in a volleyball service test in rested and physical exertion conditions in elite and near-elite adolescent players

Volleyball coaches use skill tests to assess the skill level of their players and to predict the players' future success. In a typical volleyball skill test, the players are asked to perform the skill after a rest period. The purpose of this study was to assess a volleyball service test performed not only under a rested condition but also immediately following physical exertion. Twenty-six male adolescent volleyball players (15 elite players of a coherent team [team A; mean age = 16.4 years] and 11 near-elite players of a high school team [team B; mean age = 16.3 years]) performed a service test in a rested condition and following physical exertion. The physical exertion consisted of a block at the net followed by a dig at the 3-m line, both performed twice, and again a block at the net. The players performed 10 consecutive serves under the rested condition and 5 sets of 2 consecutive serves under the physical exertion condition. The points for each serve were allotted according to predesignated target areas. The data analyses indicated no differences between the teams in service performances. No differences between the players' service scores in rested and physical exertion conditions were found. A high correlation (r = 0.97) was obtained between the total score of the test and the number of successful 7-point serves. A moderate correlation (r = 0.69) was found for the 7-point serves score following exercise. It was concluded that the number of serves hit successfully at the 7-point areas can be used by coaches as the total score of the test. In addition, the number of successful 7-point serves performed after physical exertion can provide coaches with relevant information on their players' serving skill level.     

Genetic and Epigenetic Dynamics of a Retrotransposon After Allopolyploidization of Wheat

Allopolyploidy, or the combination of two or more distinct genomes in one nucleus, is usually accompanied by radical genomic changes involving transposable elements (TEs). The dynamics of TEs after an allopolyploidization event are poorly understood. In this study, we analyzed the methylation state and genetic rearrangements of a high copied, newly amplified terminal-repeat retrotransposon in miniature (TRIM) family in wheat termed Veju. We found that Veju insertion sites underwent massive methylation changes in the first four generations of a newly formed wheat allohexaploid. Hypomethylation or hypermethylation occurred in ∼43% of the tested insertion sites; while hypomethylation was significantly predominant in the first three generations of the newly formed allohexaploid, hypermethylation became predominant in the subsequent generation. In addition, we determined that the methylation state of Veju long terminal repeats (LTRs) might be correlated with the deletion and/or insertion of the TE. While most of the methylation changes and deletions of Veju occurred in the first generation of the newly formed allohexaploid, most Veju insertions were seen in the second generation. Finally, using quantitative PCR, we quantitatively assessed the genome composition of Veju in the newly formed allohexaploid and found that up to 50% of Veju LTRs were deleted in the first generation. Retrotransposition bursts in subsequent generations, however, led to increases in Veju elements. In light of these findings, the underlying mechanisms of TRIM rearrangements are discussed.TRANSPOSABLE elements (TEs) are DNA sequences that range in size from several hundred base pairs to >15 kb and that have the ability to move to different locations within the genome. TE movement occurs through either a copy-and-paste mechanism involving RNA intermediates (class 1) or a cut-and-paste mechanism involving DNA intermediates (class 2). Class 1 elements are also called retrotransposons, or retroelements, and comprise two main types: (1) long terminal repeat (LTR) retrotransposons, flanked by LTRs, and (2) non-LTR elements (such as long interspersed nuclear elements and short interspersed nuclear elements).LTR retrotransposons are the most abundant mobile elements in plant genomes (Feschotte et al. 2002), as the replicative mode of retroelement transposition enables the LTR retrotransposon to accrue in high copy number. Indeed, in some grasses, LTR retrotransposons represent up to 90% of the genome (Bennetzen and Kellogg 1997; Feschotte et al. 2002). As such, retrotransposon sequences function well as substrates for illegitimate and unequal recombinations that can lead to a variety of mutations, such as deletions, insertions, translocations, and others (Parisod et al. 2009).The replicative nature of TEs seems to be stimulated by a variety of specific stress conditions (reviewed by Wessler 1996; Capy et al. 2000; Grandbastien et al. 2005), including challenges to the genome such as interspecific hybridization, an idea first proposed by Barbara McClintock 26 years ago (McClintock 1984). Accordingly, allopolyploidization is usually coupled with rapid and reproducible genomic changes, including the elimination of DNA sequences (Liu et al. 1998a,b; Ozkan et al. 2001; Shaked et al. 2001; Adams and Wendel 2005b; Skalicka et al. 2005), gene silencing (Chen and Pikaard 1997; Comai et al. 2000; Kashkush et al. 2002; Simons et al. 2006), alteration of cytosine methylation (Shaked et al. 2001; Madlung et al. 2002; Salmon et al. 2005; Beaulieu et al. 2009; Xu et al. 2009), activation of genes and retrotransposons (Kashkush et al. 2002, 2003; O''Neill et al. 2002), massively altered gene expression patterns (Kashkush et al. 2002; Wang et al. 2006), and organ-specific subfunctionalization, i.e., differential expression of homeoalleles in different tissues and at different developmental stages (Adams et al. 2003; Adams and Wendel 2004). These and other studies (Levy and Feldman 2002; Osborn et al. 2003; Adams and Wendel 2005a; Rapp and Wendel 2005; Chen and Ni 2006; Chen 2007) demonstrate the dynamic nature of allopolyploid plant genomes.Although allopolyploidization has generally been assumed to induce large bursts of TE activity (Matzke and Matzke 1998), several studies that focused on different allopolyploid systems failed to provide any evidence for a transposition burst and offered only limited evidence for the transposition of specific TEs (Madlung et al. 2005; Ainouche et al. 2009; Beaulieu et al. 2009). In newly formed Arabidopsis allopolyploids, no evidence for transposition bursts was reported (Beaulieu et al. 2009), although limited evidence suggested that transposition events occurred in a specific TE called Sunfish (Madlung et al. 2005). Little evidence of TE transposition was found in a natural population of the 150-year-old allopolyploid, Spartina anglica (Ainouche et al. 2009), and no evidence of transposition of Wis 2-1A retrotransposons in a newly formed wheat allotetraploid was present (Kashkush et al. 2003). The results of these works and others indicate that, in the short term, TE proliferation after allopolyploidization may be restricted to a few specific TEs in particular allopolyploidy systems (Parisod et al. 2009).This study entailed a detailed investigation of the methylation patterns and rearrangements of a one terminal-repeat retrotransposon in miniature (TRIM) family in wheat termed Veju. TRIM elements possess the classical structure of LTR retrotransposons, but they are distinguished by their small overall sizes (0.4 to ∼2.5 kb). A nonautonomous retrotransposon, Veju is 2520 bp long with 374 bp of identical LTRs, yet does not contain the proteins required for retrotransposition (Sanmiguel et al. 2002). However, because Veju elements contain polypurine tracts (PPTs) and primer binding sites (PBSs), they are capable of transposing if the retrotransposition proteins are available from another source. In addition, the identical sequences of the Veju 5′ and 3′ LTRs indicate that some members of the Veju family retain retrotransposition activity.In silico analysis of Veju sequences revealed them to be one of the most active and most recently inserted sequences in the wheat genome (Sanmiguel et al. 2002; Sabot et al. 2005a). As such, we have determined and compared the methylation patterns of >880 Veju insertion sites in the first four generations of a newly formed wheat allohexaploid, as well as in the parental lines. We then tested the correlation between the cytosine methylation and genetic rearrangements (i.e., deletions and insertions) of Veju and addressed the precise developmental timing of these rearrangements. Finally, we successfully tested overall changes in the copy numbers of Veju in the newly formed allohexaploid using real-time quantitative PCR.