A microtitre plate assay for measuring glycosidase activity

Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.     

Reovirus infection and tissue injury in the mouse central nervous system are associated with apoptosis.

Reovirus serotype 3 strains infect neurons within specific regions of the neonatal mouse brain and produce a lethal meningoencephalitis. Viral replication and pathology colocalize and have a predilection for the cortex, hippocampus, and thalamus. We have shown previously that infection of cultured fibroblasts and epithelial cells with reovirus type 3 Dearing (T3D) and other type 3 reovirus strains results in apoptotic cell death, suggesting that apoptosis is a mechanism of cell death in vivo. We now report that T3D induces apoptosis in infected mouse brain tissue. To determine whether reovirus induces apoptosis in neural tissues, newborn mice were inoculated intracerebrally with T3D, and at various times after inoculation, brain tissue was assayed for viral antigen by immunostaining and apoptosis was identified by DNA oligonucleosomal laddering and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cells were also stained with cresyl violet to detect morphological changes characteristic of apoptosis, including chromatin condensation and cell shrinkage. DNA laddering was detected in T3D- but not in mock-infected brain tissue. Apoptotic cells were restricted to the same regions of the brain in which infected cells and tissue damage were observed. These findings suggest that virus-induced apoptosis is a mechanism of cell death, tissue injury, and mortality in reovirus-infected mice. The correlation between apoptosis and pathogenesis in vivo identifies apoptosis as a potential target for molecular and pharmacological strategies designed to curtail or prevent diseases resulting from induction of this cell death pathway.     

Solvent cage effects: the influence of radical mass and volume on the recombination dynamics of radical cage pairs generated by photolysis of [CpCH2CH2N(CH3)C(O)(CH2)nCH3Mo(CO)3]2 (n = 3, 8, 13, 18) (Cp = eta 5-C5H4) complexes

The photochemistry of the [(CpR)Mo(CO)(3)](2) molecules, where CpR = eta(5)-C(5)H(4)(CH(2))(2)C(O)NCH(3)(CH(2))(n)CH(3) (n = 3, 8, 13, and 18), was examined using femtosecond pump-probe transient absorption spectroscopy. The goal of this study was to investigate the importance of radical size and mass on the dynamics and efficiency of geminate radical-radical recombination. The femtosecond results demonstrated the lack of any size/mass dependence of the recombination efficiency. This finding contrasts with results from a prior study that did find a size/mass dependence using a steady-state photochemical technique. To explain these conflicting results, it is proposed that the femtosecond pump-probe results are a measurement of the efficiency of primary geminate recombination whereas the steady-state method results are a measurement of the sum of primary and secondary geminate recombination efficiencies. The size/mass dependence is evident in the latter because secondary geminate recombination is a slower diffusive recombination process and therefore depends on the steric properties of the radicals. Although the existence of primary and secondary recombination channels is often taken for granted, experimental differentiation of primary and secondary caging has proven to be difficult because it is not possible for a single experimental technique to span the entire timescale for recombination of a radical cage pair and adequately resolve these recombination pathways.     

RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER-containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Oil Spill Response Risk Judgments,Decisions, and Mental Models: Findings from Surveying U.S. Stakeholders and Coastal Residents

This study applies a mental models survey approach to assess public thinking about oil spills and oil spill response. Based on prior interdisciplinary oil spill response research, the study first applies qualitative interview results and a response risk decision model to the design of a survey instrument. The decision model considers controlled burning, public health, and seafood safety. Surveying U.S. coastal residents (36,978 pairs of responses) through Google Insights identifies beliefs and gaps in understanding as well as related values and preferences about oil spills, and oil spill responses. A majority of respondents are concerned about economic impacts of major oil spills, and tend to see ocean ecosystems as fragile. They tend to see information about chemical dispersants as more important than ecological baseline information, and dispersants as toxic, persistent, and less effective than other response options. Although respondents regard laboratory studies as predictive of the effects of oil and of controlled burning, they are less confident that scientists agree on the toxicity and effectiveness of dispersants. The results illustrate opportunities to reframe discussions of oil spill response in terms of tradeoffs between response options, and new possibilities for assessing public opinions and beliefs during events.     

Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment). An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8). A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20–40% for cells of older passage (6–8 passages) whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.     

Spatial refuges and associational defenses promote harmful blooms of the alga Caulerpa sertularioides onto coral reefs

Extreme population fluctuations, or outbreaks, are driven by interacting processes that are often more complex than isolated changes in consumer or resource control. Blooms of the macroalga Caulerpa sertularioides in the eastern tropical Pacific overgrew and killed reef-building corals, with blooms onto reefs corresponding to cool La Niña phases of inter-decadal fluctuations of the El Niño–Southern Oscillation. We quantified factors responsible for the maintenance of C. sertularioides patches in off-reef areas, namely an associational mutualism with an epiphytic cyanobacteria (Lyngbya majuscula), coupled with spatial refuges at the scales of individual thalli and habitat. Maintenance of near reef algal populations with a strong response to nutrient addition showed that these populations were primed to bloom onto reefs in response to enhanced nutrient delivery, such as those potentially associated with La Niña conditions. However, our experiments demonstrated that no single factor related to consumer or resource control was likely to stimulate bloom formation in isolation. Rather, we propose a novel model of reef bloom formation where off-reef blooms are sustained by processes reducing consumer control, and then bloom onto reefs through an interaction between increased allochthonous nutrient input and an uncoupling of consumer control by an association with epiphytic cyanobacteria.