A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

Ancient drainages divide cryptic species in Australia's arid zone: morphological and multi-gene evidence for four new species of Beaked Geckos (Rhynchoedura)

Deserts and other arid zones remain among the least studied biomes on Earth. Emerging genetic patterns of arid-distributed biota suggest a strong link between diversification history and both the onset of aridification and more recent cycles of severe aridification. A previous study based on 1 kb of mtDNA of the monotypic gecko genus Rhynchoedura identified five allopatric clades across the vast Australian arid zone. We supplemented this data with 2.2kb from three nuclear loci and additional mtDNA sequences. Phylogenetic relationships estimated from the mtDNA data with ML and Bayesian methods were largely concordant with relationships estimated with the nDNA data only, and mtDNA and nDNA data combined. These analyses, and coalescent-based species-tree inference methods implemented with (?)BEAST, largely resolve the relationships among them. We also carried out an examination of 19 morphological characters for 268 museum specimens from across Australia, including all 197 animals for which we sequenced mtDNA. The mtDNA clades differ subtly in a number of morphological features, and we describe three of them as new species, raise a fourth from synonymy, and redescribe it and the type species, Rhynchoedura ornata. We also describe a morphologically distinctive new species from Queensland based on very few specimens. The distribution of arid zone clades across what is now relatively homogeneous sand deserts seems to be related to a topographic divide between the western uplands and eastern lowlands, with species' distributions correlated with dryland rivers and major drainage divides. The existence of five cryptic species within the formerly monotypic Rhynchoedura points to ancient divergences within the arid zone that likely were driven by wet phases as well as dry ones.     

T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies THEMIS as a new TCR signalosome component

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.     

Growth control of human foreskin fibroblasts and inhibition of extracellular sialidase activity by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid

Normal human fibroblasts have two extreme modes of existence in culture, quiescent and proliferative. The growth and division of these cells are usually well regulated by the action of various endogenously generated stimulators and inhibitors. We have speculated that an extracellular sialidase may be involved in the regulation of growth and that inhibition of this activity might decrease or abolish cell growth. To test this hypothesis, we have incubated preconfluent cultures of fibroblasts in the presence and absence of a potent sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Treatment of cells with this inhibitor resulted in the inhibition of an extracellular sialidase activity for up to 24 h and had a marked growth inhibitory effect in a concentration-dependent manner. The effect of the inhibitor on cell proliferation was specific and reversible. During a chase period of 48 h after pulse labeling cells with [3H]N-acetylmannosamine and [14C]serine, there was a 15% decrease of [3H]sialic acid in the membrane-bound GM3 with 80 microM inhibitor in the medium, as compared with a 32% decrease in the controls. Our results suggest that an extracellular sialidase may participate in cell-surface modifications that accompany (or control) the changes observed when cells traverse the cell cycle, from the quiescent to the proliferative phase.     

Substituted Sialic Acid Prosthetic Groups as Determinants of Viral Hemagglutination

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.