Coupling of agonist binding to effector domain activation in metabotropic glutamate-like receptors

Many membrane receptors are made of a ligand binding domain and an effector domain mediating intracellular signaling. This is the case for the metabotropic glutamate-like G-protein-coupled receptors. How ligand binding leads to the active conformation of the effector domain in such receptors is largely unknown. Here, we used an evolutionary trace analysis and mutagenesis to identify critical residues involved in the allosteric coupling between the Venus flytrap ligand binding domain (VFT) and the heptahelical G-protein activating domain of the metabotropic glutamate-like receptors. We have shown that a conserved interdomain disulfide bridge is required for this allosteric interaction. Taking into account that these receptors are homodimers, this finding provides important new information explaining how the different conformations of the dimer of VFT lead to different signaling of such dimeric receptors.     

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.     

Desirable plant root traits for protecting natural and engineered slopes against landslides

Slope stability models traditionally use simple indicators of root system structure and strength when vegetation is included as a factor. However, additional root system traits should be considered when managing vegetated slopes to avoid shallow substrate mass movement. Traits including root distribution, length, orientation and diameter are recognized as influencing soil fixation, but do not consider the spatial and temporal dimensions of roots within a system. Thick roots act like soil nails on slopes and the spatial position of these thick roots determines the arrangement of the associated thin roots. Thin roots act in tension during failure on slopes and if they traverse the potential shear zone, provide a major contribution in protecting against landslides. We discuss how root traits change depending on ontogeny and climate, how traits are affected by the local soil environment and the types of plastic responses expressed by the plant. How a landslide engineer can use this information when considering slope stability and management strategies is discussed, along with perspectives for future research. This review encompasses many ideas, data and concepts presented at the Second International Conference ‘Ground Bio- and Eco-engineering: The Use of Vegetation to Improve Slope Stability—ICGBE2’ held at Beijing, China, 14–18 July 2008. Several papers from this conference are published in this edition of Plant and Soil.     

A qualitative analysis of emotional effector patterns and their feedback

This paper is devoted to the study of the relationship between the subjective component (feelings) and the behavioral aspect of emotions. The following emotions were studied: fear-anxiety, anger-aggression, joy-laughter, love-eroticism, love-tenderness, and sadness-tears. The observations were performed with three different groups of people: patients with anxiety neurosis, students under hypnosis, and drama students. Each emotion was characterized by a specific set of reactions in the respiratory pattern, heart activity, muscular activity, and facial expression. The feelings were correlated with the behavioral patterns and each time the behavioral patterns were interfered with a concomitant modification of the subjectivity component was observed. The direct performance of the behavioral emotional patterns in the absence of the emotogenic stimulus produced the feeling corresponding to the mimicked emotion. If the subjects were stimulated with an emotogenic stimulus during the direct performance of the behavioral patterns of another emotion, they confessed to have the feeling corresponding to the mimicked emotion, and not to the emotion belonging to the emotogenic stimulus. The role played by the feedback from the effector organs in the determination of the subjective emotional states is discussed.     

Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.     

"In vivo" intraneuronal trafficking of G protein coupled receptors in the striatum: regulation by dopaminergic and cholinergic environment

We have studied "in vivo" neurochemically identified striatal neurons to analyze the localisation and the trafficking of dopamine and acetylcholine G protein coupled receptors (GPCR) (D1R, D2R, m2R and m4R) under the influence of neurotransmitter environment. We have identified receptors in tissue sections through immunohistochemical detection at the light and electron microscopic level. We have identified receptors in normal animals and after acute and chronic stimulations. We have quantified receptors through image analysis at the electron microscopic level in relation to various subcellular compartments. Our results demonstrate that, in normal conditions, GPCRs are mostly associated with plasma membrane of the striatal neurons, mostly at extra-synaptic sites. In certain instances (m4R; D2R), receptors have prominent localisation inside the rough endoplasmic reticulum. Our results also show that two distinct receptors for a same neurotransmitter may have distinct subcellular localisation in a same neuronal population (m2R versus m4R) and that the same neurotransmitter receptor (m4R) can have distinct localisation in distinct neuronal populations (cytoplasm versus cell surface). After acute stimulation, cell surface receptors undergo dramatic subcellular changes that involve plasma membrane depletion, internalisation in endosomes and in multivesicular bodies. Such changes are reversible after the end of the stimulation and are blocked by antagonist action. Chronic stimulation also provokes changes in subcellular localisation with specific pattern: plasma membrane depletion, and exaggerated storage of receptors in rough endoplasmic reticulum and eventually Golgi complex (D1R; m2R and m4R). Decreasing chronic receptor stimulation reverses such changes. These results demonstrate that, "in vivo", in the striatum, GPCRs undergo complex intraneuronal trafficking under the influence of neurochemical environment in conditions that dramatically modulate the number of cell surface receptors available for interaction with neurotransmitters or drugs. This confirms that "in vivo", the trafficking and the subcellular compartmentalization of GPCRs may contribute to regulate neuronal sensitivity and neuronal interactions in physiological, experimental and pathological conditions, including in therapeutic conditions.     

hMutS alpha is protected from ubiquitin-proteasome-dependent degradation by atypical protein kinase C zeta phosphorylation

The hMutS alpha (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutS alpha proteins were degraded by the ubiquitin-proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutS alpha protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKC zeta regulates ubiquitination, degradation, and levels of hMutS alpha proteins. Using both PKC zeta-transfected U937 and PKC zeta siRNA-transfected MRC-5 cell lines, we found that PKC zeta protein expression was correlated with that of hMutS alpha as well as with MMR activity, but was inversely correlated with hMutS alpha protein ubiquitination and degradation. Interestingly, PKC zeta interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCzeta mediates phosphorylation events decreasing hMutS alpha protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKC zeta modulates hMutS alpha stability and protein levels, and suggest a role for PKC zeta in genome stability by regulating MMR activity.