Lipid ordering and enzymic activities in chromaffin granule membranes.

1. The interaction of a variety of fluorescent probes with the membranes of adrenal medullary chromaffin granules is described. 2. Changes in the motional properties of the bound probes with temperature were investigated and evidence is presented which indicates that ordering of the membrane lipids occurs below 33 degrees C. 3. The ordering is characteristics of the membrane lipids and is retained by sonicated aqueous dispersions of the total lipid extracted from chromaffin granule membranes. 4. The ATPase and NADH:acceptor oxidoreductase activities of the chromaffin granule membrane have discontinuous Arrhenius temperature versus activity relationships with 'transitions' at 33 degrees C. 5. The ATPase has a second transition at 36.5 degrees C. 6. The 33 degrees C 'transition' for the NADH:acceptor oxidoreductase is removed by treatment with the detergent Triton X-100. 7. The correlation between the onset of lipid ordering and the change in activation energy of the membrane-bound enzyme activities is discussed in terms of the co-operative interactions of the different membrane components. The possible role of lipid ordering in exocytosis is discussed.     

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.     

Multifactor-dimensionality reduction reveals high-order interactions among estrogen-metabolism genes in sporadic breast cancer

One of the greatest challenges facing human geneticists is the identification and characterization of susceptibility genes for common complex multifactorial human diseases. This challenge is partly due to the limitations of parametric-statistical methods for detection of gene effects that are dependent solely or partially on interactions with other genes and with environmental exposures. We introduce multifactor-dimensionality reduction (MDR) as a method for reducing the dimensionality of multilocus information, to improve the identification of polymorphism combinations associated with disease risk. The MDR method is nonparametric (i.e., no hypothesis about the value of a statistical parameter is made), is model-free (i.e., it assumes no particular inheritance model), and is directly applicable to case-control and discordant-sib-pair studies. Using simulated case-control data, we demonstrate that MDR has reasonable power to identify interactions among two or more loci in relatively small samples. When it was applied to a sporadic breast cancer case-control data set, in the absence of any statistically significant independent main effects, MDR identified a statistically significant high-order interaction among four polymorphisms from three different estrogen-metabolism genes. To our knowledge, this is the first report of a four-locus interaction associated with a common complex multifactorial disease.     

Contrasting effects of plant richness and composition on insect communities: a field experiment

We experimentally separated the effects of two components of plant diversity-plant species richness and plant functional group richness-on insect communities. Plant species richness and plant functional group richness had contrasting effects on insect abundances, a result we attributed to three factors. First, lower insect abundances at higher plant functional group richness were explained by a sampling effect, which was caused by the increasing likelihood that one low-quality group, C4 grasses, would be present and reduce average insect abundances by 25%. Second, plant biomass, which was positively related to plant functional group richness, had a strong, positive effect on insect abundances. Third, a positive effect of plant species richness on insect abundances may have been caused by greater availability of alternate plant resources or greater vegetational structure. In addition, a greater diversity of insect species, whose individual abundances were often unaffected by changes in plant species richness, may have generated higher total community abundances. After controlling for the strong, positive influence of insect abundance on insect diversity through rarefaction, insect species richness increased as plant species richness and plant functional group richness increased. Although these variables did not explain a high proportion of variation individually, plant species richness and plant functional group richness had similar effects on insect diversity and opposing effects on insect abundances, and both factors may explain how the loss of plant diversity influences higher trophic levels.     

Flavivirus isolations from mosquitoes collected from Saibai Island in the Torres Strait, Australia, during an incursion of Japanese encephalitis virus

Adult mosquitoes (Diptera: Culicidae) were collected in January and February 2000 from Saibai Island in the Torres Strait of northern Australia, and processed for arbovirus isolation during a period of Japanese encephalitis (JE) virus activity on nearby Badu Island. A total of 84 210 mosquitoes were processed for virus isolation, yielding six flavivirus isolates. Viruses obtained were single isolates of JE and Kokobera (KOK) and four of Kunjin (KUN). All virus isolates were from members of the Culex sitiens Weidemann subgroup, which comprised 53.1% of mosquitoes processed. Nucleotide sequencing and phylogenetic analysis of the pre-membrane region of the genome of JE isolate TS5313 indicated that it was closely related to other isolates from a sentinel pig and a pool of Cx. gelidus Theobald from Badu Island during the same period. Also molecular analyses of part of the envelope gene of KUN virus isolates showed that they were closely related to other KUN virus strains from Cape York Peninsula. The results indicate that flaviviruses are dynamic in the area, and suggest patterns of movement south from New Guinea and north from the Australian mainland.     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp