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This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled.
Atlantic mollies (Poecilia mexicana) inhabit a variety of surface habitats, but they also occur in a sulfur cave in southern Mexico. We examined male mate choice relative to female body size in the cave population and in the most closely related surface-dwelling population from a nearby river. Males from both populations were either light- or dark-reared and could choose between two differently sized females either on the basis of visual cues in light or on the basis of solely nonvisual cues in darkness. Sexual preferences were estimated from the degree of association. Cave molly males always showed a preference for the larger female, both in light and in darkness. Among the surface males, only light-reared males showed a preference in the visual cues test, but not in darkness. In a control experiment, we demonstrated that male association preferences directly translate into actual mating preferences. Apparently, using visual cues for mate choice is the ancestral state in this system, and using nonvisual cues has evolved as a novel trait in the cave population. We discuss the evolution of nonvisual male mate choice in the context of changed environmental conditions, namely the absence of light, hypoxia, and toxic hydrogen sulfide in the cave
We summarize here the development of various piezoelectric biosensors utilizing cholinesterase (ChE) as the recognition element. In our work we studied the interaction between cholinesterase and its ligands (propidium, carnitine, benzylgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) and paraoxon). The sensor modification was based on a self-assembled monolayer (SAM) of a thiol compound (11-mercaptoundecanoic acid) on the gold electrode and the subsequent covalent coupling of the cholinesterase ligand to this SAM. The ligand-modified piezoelectric sensors were placed in a flow system to allow the on-line monitoring of cholinesterase binding and the enzymatic activity quantification by amperometry. Cholinesterases from different species-acetylcholinesterase (AChE) from Electrophorus electricus , AChE from Drosophila melanogaster , and butyrylcholinesterase (BChE) of human origin-were tested on the various immobilized ligands. Our research allowed the development of a competitive assay for the detection of organophosphates in river water samples using the BZE-DADOO-modified piezosensor. Another direction of research was pointed on the characterization of the interactions between ChE and its ligands. The kinetic binding constants were derived using a one- to-one binding model
Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. in the blowfly (Calliphora vicina) salivary gland, V- ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the CAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that CAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V, components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus
Calpain 1-gamma filamin interaction in muscle cells :a possible in situ regulation by PKC-alpha
(2006)
Calpain 1-gamma filamin interaction in muscle cells : a possible in situ regulation by PKC-alpha
(2006)
Calpain 1-gamma filamin interaction in muscle cells : a possible in situ regulation by PKC-alpha
(2006)
Calpains are a family of calcium-dependant cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain I in the muscle contractile apparatus and specifically in the N1- and N2-fines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin - a specific muscle isoform of the filamin family - is a calpain, I substrate and to characterise this interaction. gamma-Filamin is a major muscle architectural protein located in the Z-fine and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain I in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain I are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C- terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain I in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.
We investigated whether female association preferences for males are influenced by black spot disease (BSD), a parasite induced change of the host phenotype. We compared three different species of fish: a gynogenetic hybrid species, Poecilia formosa (amazon molly) and two sexual species (Poecilia latipinna and Poecilia mexicana), which were involved in the natural hybridisation leading to the amazon molly. Contrary to their sexual relatives, asexual amazon mollies significantly avoided images of males infected with black spot disease. We propose that amazon molly females have direct fitness benefits from choosing healthy males. The adaptive significance of the preference for BSD-uninfected males in the asexual amazon molly is yet unclear but may involve avoidance of predation or parasite infection as well as increased sperm availability
Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)
(2006)
A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).