Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

Nitrogen Oxyanion-dependent Dissociation of a Two-component Complex That Regulates Bacterial Nitrate Assimilation

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.     

A unifying theory for methods of systematic analysis

A unifying theory for systematic analysis states that a number of methods should be used jointly to cope with various kinds of data; also that groups should be as consistent as possible, be made with least information loss, and where needed, be polythetic. A test of relationship, homogeneity, can use various kinds of data. It can take account of the internal variation of aggregate items such as genera. It can give due emphasis to smaller clusters that have likely important contexts of external items. It helps in analysing trends, cores and hazes in dendrograms. A proposed detector for formal groups can be based on measures of isolation, identifiability and inclusiveness. Non-mathematical, inter-item reaction tests such as hybridization and serology can also be used in grouping. All relationship data are used polythetically to reveal natural groups. This leads to a unified informational concept for taxa. This is more useful than the biological species concept that is restricted to inter-breeding data. All the methods appear to be analogues of the powerful human grouping instinct. The resulting compatibility is important as precise methods are needed mainly when the data are too complex for the mind to use reliably. Cladograms can be made by self-graded deweighting of homogeneity and agglomerative clustering. Unlike classical cladistics this can reveal any polythetic group. Finding the derived states for making cladograms is often much too hypothetical for a fully cladistic approach to be properly precise. Instead, where the evidence is weak, a milder strength of graded deweighting is used for the cladistic properties, which help to show relationships along with the others. Axiomatic failures of other classes of grouping methods are discussed. Unavoidable remnants of instinctive processing lower the precision of all the methods. The Uniter computer program, based on the theory, is tested with finely graded values of artificially ‘evolved’ items and with coarsely coded cladistic data. The results show that with natural data, the program should act as a fairly sensitive probe of past evolutionary branching. Another test shows how specimens from species complexes can be grouped and how distinctions between groups are analysed.     

Electron donation in Photosystem II

The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D⁺ (Signal II) and Q⁻_A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D⁺. The donor D was then shown to be oxidized in the dark by the S₂ state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.     

Stonefly nymphs use hydrodynamic cues to discriminate between prey

Summary Playback experiments conducted in a Rocky Mountain, USA, stream determined whether predatory stonefly nymphs (Kogotus modestus; Plecoptera: PerlodiMae) used hydrodynamic cues to discriminate prey species from nonprey species. In the laboratory we recorded pressure wave patterns associated with swimming escape behavior of Baetis bicaudatus (Baetidae), the favored mayfly prey species, and those of a nonprey mayfly, Ephemerella infrequens (Ephemerellidae). We video taped the responses of 24-h starved Kogotus to Baetis playbacks, Ephemerella playbacks or no playbacks made by oscillating (or not) live mayflies (Ephemerella) or clear plastic models placed within in situ flow-through observation boxes. The probability of attacks per encounter with Baetis playbacks was highest and independent of the model type used, but Kogotus also showed an unexpected high probability of attacks per encounter when Ephemerella playbacks were made through live Ephemerella. Thus, Kogotus discriminated between Baetis and Ephemerella swimming patterns but only when playbacks were made through the plastic model. Kogotus never attacked motionless mayflies or motionless plastic models. We allowed some Kogotus to successfully capture one small Baetis immediately before playbacks, which resulted in a much higher probability of attacks per encounter with Baetis playbacks on either model and a heightened discrimination of prey versus nonprey playbacks. The probability of attacks per encounter by Kogotus with live Baetis swimming under similar experimental conditions was strikingly similar to its response to Baetis playbacks made by oscillating the plastic model after a successful capture. Order of playback presentation (Baetis first or Ephemerella first) did not influence predatory responses to mayfly swimming patterns. This study is the first to document the use of hydrodynamic cues by stream-dwelling predators for discrimination of prey from nonprey and provides a mechanism to explain selective predation by stoneflies on Baetis in nature.