Structural Insights into Serine-rich Fimbriae from Gram-positive Bacteria

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.     

Cannibalistic Interactions Resulting from Indiscriminate Predatory Behavior in Tadpoles of Poison Frogs (Anura: Dendrobatidae)1

Poison frogs in the genus Dendrobates have very small clutch sizes (2–6 eggs among species for which there are data) and typically transport their tadpoles singly to small phytotelmata, such as bromeliad tanks, leaf axils, fallen fruit capsules, and treeholes. Tadpoles of many species are predaceous, consuming larvae of insects that use the same microhabitat for breeding, such as giant damselflies and mosquitoes. Previous studies and observations on the behavior of poison frog tadpoles led us to question whether tadpoles might be cannibalistic. We studied a population of Dendrobates castaneoticus in lowland rainforest in Pará, Brazil; additional data were collected on Dendrobates auratus in Nicaragua. At the study site in Brazil, we established a grid of 40 Brazil nut capsules, the microhabitat used by D. castaneoticus for tadpole deposition. Of 42 tadpoles deposited during the 55 days of the study, 20 were killed or died; 16 of these were presumably killed by conspecific tadpoles. Growth rate and time to metamorphosis was higher among tadpoles that consumed three or more tadpoles or relatively large larvae of the mosquito Trichoprosopon digitatum, a colonist of newly opened Brazil nut capsules. We propose that selection has favored the development of predatory behavior in poison frog tadpoles primarily as a mechanism to eliminate predators from the small phytotelmata in which they develop and that cannibalism is a secondary outcome of this behavior. Predatory behavior also provides tadpoles with a source of food, which is frequently limited in these microhabitats. Additional studies of the biology of tadpoles of other species of Dendrobates are needed to determine the evolution of predatory and cannibalistic behavior in the clade.     

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

Synapse-specific downregulation of NMDA receptors by early experience: a critical period for plasticity of sensory input to olfactory cortex

Olfaction is required at birth for survival; however, little is known about the maturation of olfactory cortical circuits. Here we show that in vivo sensory experience mediates the development of excitatory transmission in pyramidal neurons of rat olfactory cortex. We find a postnatal critical period during which there is an experience-dependent increase in the contribution of AMPARs versus NMDARs to transmission at primary sensory synapses but not associational inputs. The shift in receptors underlying transmission is mediated by a strong activity-dependent downregulation of NMDARs and modest increase in AMPARs. Sensory activity leads to a loss of "silent" NMDAR-only synapses and an increase in threshold for inducing long-term plasticity. These results indicate the importance of early olfactory experience in the establishment of cortical circuits and could reflect mechanisms governing early olfactory "imprinting."     

Fusogenic activity of EFF-1 is regulated via dynamic localization in fusing somatic cells of C. elegans

BACKGROUND: Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS: We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS: EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.