Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.     

Pollination Ecophysiology of Mercurialis annua L. (Euphorbiaceae), an Anemophilous Species Flowering all Year Round

Mercurialis annua L. is a dioecious anemophilous species thatflowers all year round in central and southern Italy. The flowersof both sexes are dimorphic: the female flower has a vestigialcalyx; the male flower consists only of a calyx that opens atanthesis. The anthers always dehisce after anthesis. The anthesisof male flowers seems to be temperature dependent, whereas antherdehiscence is related to relative humidity. The pollen grainsvary in volume according to the season: they are smaller whenrelative humidity is low and vice versa. They always decreasein volume after anther dehiscence and have the capacity to varyin volume and reach equilibrium with a changing environment.Viability is high, but may drop suddenly during heavy rain orhail that damage the exposed male flowers. The number of pollengrains per stigma varies from 0 to 300. The data is discussedin relation to the type of pollination and environmental characteristics.Copyright1994, 1999 Academic Press Mercurialis annua, dioecism, anthesis, anther dehiscence, pollen volume, pollen viability, anemophilous pollination, pollination ecology     

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.     

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.     

Rethinking the evolution of extant sub‐Saharan African suids (Suidae,Artiodactyla)

Gongora, J., Cuddahee, R. E., do Nascimento, F. F., Palgrave, C. J., Lowden, S., Ho, S. Y. W., Simond, D., Damayanti, C. S., White, D. J., Tay, W. T., Randi, E., Klingel, H., Rodrigues‐Zarate, C. J., Allen, K., Moran, C. & Larson, G. (2011). Rethinking the evolution of extant sub‐Saharan African suids (Suidae, Artiodactyla). —Zoologica Scripta, 40, 327–335. Although African suids have been of scientific interest for over two centuries, their origin, evolution, phylogeography and phylogenetic relationships remain contentious. There has been a long‐running debate concerning the evolution of pigs and hogs (Suidae), particularly regarding the phylogenetic relationships among extant Eurasian and African species of the subfamily Suinae. To investigate these issues, we analysed the mitochondrial and nuclear DNA sequences of extant genera of Suidae from Eurasia and Africa. Molecular phylogenetic analyses revealed that all extant sub‐Saharan African genera form a monophyletic clade separate from Eurasian suid genera, contradicting previous attempts to resolve the Suidae phylogeny. Two major sub‐Saharan African clades were identified, with Hylochoerus and Phacochoerus grouping together as a sister clade to Potamochoerus. In addition, we find that the ancestors of extant African suids may have evolved separately from the ancestors of modern day Sus and Porcula in Eurasia before they colonised Africa. Our results provide a revision of the intergeneric relationships within the family Suidae.     

Breeding systems in two species of the genus Helleborus (Ranunculaceae)

ABSTRACT

The results of various types of manual and free pollination are reported in Helleborus bocconei and Helleborus foetidus, two sympatric species of the understorey of submediterranean woods that flower in January–March and are pollinated by bumble-bees of the genus Bombus. Both species were found to be partially self-compatible even if at different rates; geitonogamy seems to be more frequent in H. foetidus and spontaneous self-pollination does not usually occur for morphological reasons, and the species are not apomictic. Efficiency in seed production after free pollination was very high indicating that in spite of harsh climatic condition, there were no limitations imposed by pollen or pollinators.     

Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.     

A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery

Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8(+) IFN-gamma and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-gamma in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30-40 residues (3-4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-gamma responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8(+) responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8(+) T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8(+) IFN-gamma and HTL responses.