Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae)

The 60 000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life‐history traits, which range from saprophagy over phytophagy to parasitism and predation. However, the direction of evolutionary change between the different modes remains unclear. Here, we use the Scathophagidae (Diptera) for reconstructing the direction of change in this relatively small family (≈ 250 spp.) whose larval habits mirror the diversity in natural history found in Cyclorrhapha. We subjected a molecular data set for 63 species (22 genera) and DNA sequences from seven genes (12S, 16S, Cytb, COI, 28S, Ef1‐alfa, Pol II) to an extensive sensitivity analysis and compare the performance of three different alignment strategies (manual, Clustal, POY). We find that the default Clustal alignment performs worst as judged by character incongruence, topological congruence and branch support. For this alignment, scoring indels as a fifth character state worsens character incongruence and topological congruence. However, manual alignment and direct optimization perform similarly well and yield near‐identical trees, although branch support is lower for the direct‐optimization trees. All three alignment techniques favor the upweighting of transversion. We furthermore confirm the independence of the concepts “node support” and “node stability” by documenting several cases of poorly supported nodes being very stable and cases of well supported nodes being unstable. We confirm the monophyly of the Scathophagidae, its two constituent subfamilies, and most genera. We demonstrate that phytophagy in the form of leaf mining is the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation has occurred while a second origin of predation is from a saprophagous ancestor. © The Willi Hennig Society 2006.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Evidence for inbreeding depression and post-pollination selection against inbreeding in the dioecious plant Silene latifolia

In many species, inbred individuals have reduced fitness. In plants with limited pollen and seed dispersal, post-pollination selection may reduce biparental inbreeding, but knowledge on the prevalence and importance of pollen competition or post-pollination selection after non-self pollination is scarce. We tested whether post-pollination selection favours less related pollen donors and reduces inbreeding in the dioecious plant Silene latifolia. We crossed 20 plants with pollen from a sibling and an unrelated male, and with a mix of both. We found significant inbreeding depression on vegetative growth, age at first flowering and total fitness (22% in males and 14% in females). In mixed pollinations, the unrelated male sired on average 57% of the offspring. The greater the paternity share of the unrelated sire, the larger the difference in relatedness of the two males to the female. The effect of genetic similarity on paternity is consistent with predictions for post-pollination selection, although paternity, at least in some crosses, may be affected by additional factors. Our data show that in plant systems with inbreeding depression, such as S. latifolia, pollen or embryo selection after multiple-donor pollination may indeed reduce inbreeding.     

A systems biology approach: new insights into fetal growth restriction using Bayesian Networks

IL-6, IGF-II and IGFBP-2 concentrations in placental lysates were previously shown to be associated with foetal growth. This study aimed to apply a Bayesian Network (BN) model in order to investigate complex dependencies among biochemical and clinical factors and fetal growth outcome. Twenty-one Intra-Uterine Growth Restricted (IUGR) and 25 Appropriate for Gestational Age (AGA) pregnancies were followed throughout pregnancy. Information was collected on maternal and gestational age, neonatal gender, previous gynaecological history. Total protein content, IGF-II, IGFBP-1, IGFBP-2, IL-6, and TNF-alpha concentrations in placental lysates were measured, and IGF-I, IGF-II, IGFBP-1, IGFBP-2 and IL-6 relative gene expression in placenta assessed. A BN and a hybrid forecasting system were implemented: BN revealed a key role of maternal age and TNF-alpha on IUGR and confirmed a close relationship among IGF-II, IL-6 and foetal growth. A relationship between duration of gestation, appropriateness for gestational age, and placental IL-6 concentration was also confirmed. Compared with other techniques, BN showed a better accuracy. Findings confirmed a major role of maternal age in addition to IGF-II, IL-6 and TNF-alpha in IUGR. A direct role of IGFBP-2 was not shown. BN confirmed to be useful in understanding the system's biology and graphically representing variable relationships and hierarchy, particularly where, as in IUGR, many interactions among predictors exist.     

Cytotoxicity of activated monocytes on endothelial cells

Unstimulated human monocytes did not express appreciable levels of cytotoxicity on normal human umbilical vein endothelial cells (EC) in a 24-48 hr TdR release assay. On activation with IFN-gamma and LPS, monocytes had appreciable cytotoxicity on EC. Monocyte cytotoxicity on EC was not dependent on the presence of contaminating lymphoid cells. Recombinant TNF, IL-1, and IL-6 as well as monocyte supernatants did not exert a cytotoxic effect on EC. Moreover, anti-TNF, anti-IL-1, and anti-IL-6 antibodies, as well as scavengers of reactive oxygen intermediates, did not affect the cytotoxicity of activated monocytes on EC. Antibodies against the beta-chain (CD18) of leukocyte integrins inhibited the adhesion and cytotoxicity of activated monocytes on EC. Pretreatment of EC with IL-1 augmented the adhesion of monocytes on EC. Normal monocytes were not cytotoxic on IL-1-pretreated EC and IL-1 treatment did not increase the susceptibility of EC to activated monocytes. Thus adhesion is necessary but not sufficient for monocyte killing of EC. Anti-alpha L (LFA-1) antibodies markedly reduced monocyte cytotoxicity on EC, although anti-alpha X (p150) antibodies had only a modest effect. Anti-alpha M (Mac-1/CR3) antibodies were intermediate inhibitors of EC killing by activated monocytes. Thus, alpha L, beta 2 (LFA-1), and, to a lesser extent, alpha M, beta 2 (Mac-1/CR3) and alpha X, beta 2 (p 150, 95) integrins are the main adhesive structures involved in the cytotoxic interaction of activated monocytes with EC. Monocyte-mediated damage of EC could play a role as a mechanism of tissue injury under conditions of local or systemic activation of mononuclear phagocytes.     

Effect of synthetic and natural protein tyrosine kinase inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls

Basipetal auxin transport along the zucchini hypocotyl requires the cell-to-cell translocation of this hormone. Several authors have demonstrated that naphthylphthalamic acid (NPA) blocks auxin efflux. It is believed that NPA binds to a regulatory protein that modulates the activity of the carrier rather than to the carrier itself. A few regulatory mechanisms have been suggested but all lack strong supporting experimental evidence. Four classes of protein tyrosine kinase inhibitors (PTKIs) have been used to investigate the NPA perception pathway. First, six synthetic tyrphostins, inhibitors of the epidermal growth factor receptor (EGF-R), were shown to displace NPA on plasmalemma fractions. The most potent compound was (3,4-dihydroxybenzylidene)-thiocyanoacetamide (tyrphostin A47). The inactive counterpart of tyrphostin A47, (4-methoxybenzylidene)-malononitrile (tyrphostin A1), was unable to displace NPA. Tyrphostins did not inhibit auxin efflux but were antagonists of the inhibition of auxin efflux by NPA. Again, tyrphostin A47 was the most effective and tyrphostin A1 was inactive. Second, the flavonoid genistein, also an EGF-R inhibitor, showed an effect on NPA binding and NPA antagonism similar to the one for the tyrphostins. Daidzein, the inactive counterpart of genistein, was ineffective in displacing NPA and in antagonizing its inhibition of auxin efflux. Two other PTKIs, curcumin and lavendustin A, displayed similar characteristics. Calmodulin antagonists, protein serine/threonine kinase inhibitors, and phosphatase inhibitors were inactive. These results suggest that the NPA binding protein may be related to mammalian tyrosine kinases and may regulate the auxin efflux carrier by phosphorylation.     

Molecular studies of chromosomal mosaicism: relative frequency of chromosome gain or loss and possible role of cell selection.

Studies of uniparental disomy and origin of nonmosaic trisomies indicate that both gain and loss of a chromosome can occur after fertilization. It is therefore of interest to determine both the relative frequency with which gain or loss can contribute to chromosomal mosaicism and whether these frequencies are influenced by selective factors. Thirty-two mosaic cases were examined with molecular markers, to try to determine which was the primary and which was the secondary cell line: 16 cases of disomy/trisomy mosaicism (5 trisomy 8, 2 trisomy 13, 1 trisomy 18, 4 trisomy 21, and 4 involving the X chromosome), 14 cases of 45,X/46,XX, and 2 cases of 45,X/47,XXX. Of the 14 cases of mosaic 45,X/46,XX, chromosome loss from a normal disomic fertilization predominated, supporting the hypothesis that 45,X might be compatible with survival only when the 45,X cell line arises relatively late in development. Most cases of disomy/trisomy mosaicism involving chromosomes 13, 18, 21, and X were also frequently associated with somatic loss of one (or more) chromosome, in these cases from a trisomic fertilization. By contrast, four of the five trisomy 8 cases were consistent with a somatic gain of a chromosome 8 during development from a normal zygote. It is possible that survival of trisomy 8 is also much more likely when the aneuploid cell line arises relatively late in development.     

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.