1H NMR Spectroscopy of pyrrolizidine alkaloids

The ¹H NMR spectra of three pyrrolizidine alkaloids of the macrocyclic diester type, retrorsine, seneciphylline and senecionine, plus their three N-oxides have been assigned. Previous ¹H NMR studies of these pyrrolizidine alkaloids have stressed the difficulties of spectral intrepretation. The results reported here will provide a useful resource for analysis of tertiary structure in these and related compounds.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Mechanism of inhibition of site-specific recombination by the Holliday junction-trapping peptide WKHYNY: insights into phage lambda integrase-mediated strand exchange

Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study. We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage lambda Integrase (Int)-mediated reactions. We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution. The peptide acts before the step affected by site affinity (saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e. Holliday junctions) while saf mutations reduce ligation of exchanged strands.Strand exchange events leading to Holliday junctions in phage lambda integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage. We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways. Thus Int-DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway.     

Local signaling by the EGF receptor

Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.     

Regulation of Protrusion Shape and Adhesion to the Substratum during Chemotactic Responses of Mammalian Carcinoma Cells

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J. E. Segallet al.,1996,Clin. Exp. Metastasis14, 61–72), we analyzed the relationship between adhesion and chemoattractant-stimulated protrusion. Exposure to EGF led to a rapid remodeling of the adhesive contacts on adherent cells, in synchrony with extension of a flat lamellipod over the substratum. EGF-stimulated lamellipods still extended in the presence of adhesion-blocking peptides or over nonadhesive surfaces. They were, however, slightly shorter and retracted rapidly under those conditions. The major protrusive structure observed on well-spread, adherent cells, after EGF stimulation was a flat broad lamellipod, whether or not in contact with the substratum, while cells in suspension showed transient protrusive activity over the entire cell surface. We conclude that the initial adhesive status of the cell conditions the shape of the outcoming protrusion. Altogether our results suggest that, although adhesive contacts are not necessary for lamellipod extension, they play a role in stabilizing the protrusion as well as in the control of its final shape and amplitude.     

Control of directionality in the DNA strand-exchange reaction catalysed by the tyrosine recombinase TnpI

In DNA site-specific recombination catalysed by tyrosine recombinases, two pairs of DNA strands are sequentially exchanged between separate duplexes and the mechanisms that confer directionality to this theoretically reversible reaction remain unclear. The tyrosine recombinase TnpI acts at the internal resolution site (IRS) of the transposon Tn4430 to resolve intermolecular transposition products. Recombination is catalysed at the IRS core sites (IR1–IR2) and is regulated by adjacent TnpI-binding motifs (DR1 and DR2). These are dispensable accessory sequences that confer resolution selectivity to the reaction by stimulating synapsis between directly repeated IRSs. Here, we show that formation of the DR1–DR2-containing synapse imposes a specific order of activation of the TnpI catalytic subunits in the complex so that the IR1-bound subunits catalyse the first strand exchange and the IR2-bound subunits the second strand exchange. This ordered pathway was demonstrated for a complete recombination reaction using a TnpI catalytic mutant (TnpI-H234L) partially defective in DNA rejoining. The presence of the DR1- and DR2-bound TnpI subunits was also found to stabilize transient recombination intermediates, further displacing the reaction equilibrium towards product formation. Implication of TnpI/IRS accessory elements in the initial architecture of the synapse and subsequent conformational changes taking place during strand exchange is discussed.