Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions.

In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.

Combining comparative sequence and genomic data to ascertain phylogenetic relationships and explore the evolution of the large GDSL-lipase family in land plants.

The GDSL-lipase gene family is a very large subfamily within the supergene family of SGNH esterases, defined by the distinct GDSL amino acid motif and several highly conserved domains. Plants retain a large number of GDSL-lipases indicating that they have acquired important functions. Yet, in planta functions have been demonstrated for only a few GDSL-lipases from diverse species. Considering that orthologs often retain equivalent functions, we determined the phylogenetic relationships between GDSL-lipases from genome-sequenced species representing bryophytes, gymnosperms, monocots, and eudicots. An unrooted phylogenetic tree was constructed from the amino acid sequences of 604 GDSL-lipases from seven species. The topology of the tree depicts two major and one minor subfamily. This division is also supported by the unique gene structure of each subfamily. Because GDSL-lipase genes of all species are present in each of the three subfamilies, we conclude that the last common ancestor of the land plants already possessed at least one ancestral GDSL-lipase gene of each subfamily. Combined gene structure and synteny analyses revealed events of segmental duplications, gene transposition, and gene degeneration in the evolution of the GDSL-lipase gene family. Furthermore, these analyses showed that independent events of intron gain and loss also contributed to the extant repertoire of the GDSL-lipase gene family. Our findings suggest that underlying many of the intron losses was a spliceosomal-mediated mechanism followed by gene conversion. Sorting the phylogenetic relationships among the members of the GDSL-lipase gene family, as depicted by the tree and supported by synteny analyses, provides a framework for extrapolation of demonstrated functional data to GDSL-lipases, whose function is yet unknown. Furthermore, function(s) associated with specific lineage(s)-enriched branches may reveal correlations between acquired and/or lost functions and speciation.

Ascorbate peroxidase gene family in tomato: its identification and characterization.

The antioxidative response, where ascorbate peroxidase (APX) is a key enzyme, is an integral part of the plant tolerance response to environmental stresses. As a first step towards the study of the physiological role and the regulation of the members of the Apx gene family, the orthologs of the stress-sensitive cultivated tomato Solanum lycopersicum cv. M82 (Slm) and of the wild salt-tolerant species S. pennellii acc. Atico (Spa) were identified by utilizing the tomato EST database, and characterized. A redundant list of 16 virtual Apx transcripts and four singleton ESTs was shown to correspond to seven genuine Apx genes. The complete tomato Apx gene family is comprised of genes encoding three cytosolic, two peroxisomal, and two chloroplastic APXs. These genes attained differential regulatory patterns in various Slm organs. More detailed study of Apx1 and Apx2 genes, that are the products of a recent gene duplication event, shows that they have already attained differential regulation within and between Slm and Spa under control and stress conditions. It is also suggested that due to lineage-specific gene duplication and lose events, intricate phylogenetic relationships exist among the members of the Apx gene families.

Lethal contractural syndrome type 3 (LCCS3) is caused by a mutation in PIP5K1C, which encodes PIPKI gamma of the phophatidylinsitol pathway.

Lethal congenital contractural syndrome (LCCS) is a severe form of arthrogryposis. To date, two autosomal recessive forms of the disease (LCCS and LCCS2) have been described and mapped to chromosomes 9q34 and 12q13, respectively. We now describe a third LCCS phenotype (LCCS3)--similar to LCCS2 yet without neurogenic bladder. Using 10K single-nucleotide-polymorphism arrays, we mapped the disease-associated gene to 8.8 Mb on chromosome 19p13. Further analysis using microsatallite markers narrowed the locus to a 3.4-Mb region harboring 120 genes. Of these genes, 30 candidates were sequenced, which identified a single homozygous mutation in PIP5K1C. PIP5K1C encodes phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIPKI gamma ), an enzyme that phophorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate (PIP(2)). We demonstrate that the mutation causes substitution of aspartic acid with asparagine at amino acid 253 (D253N), abrogating the kinase activity of PIPKI gamma . Thus, a defect in the phosphatidylinositol pathway leading to a decrease in synthesis of PIP(2), a molecule active in endocytosis of synaptic vesicle proteins, culminates in lethal congenital arthrogryposis.

Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger.

Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-d-glucamine. A key question that has remained unsolved is how despite this, Li(+) catalyzes calcium exchange in mammalian tissues. Here we report that a Na(+)/Ca(2+) exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li(+)/Ca(2+) exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70- and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K(+)-independent exchanger that catalyzes Ca(2+) flux at a rate comparable with NCX1 but without promoting Na(+)/Ba(2+) exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li(+) promote Ca(2+) exchange whereas the known exchangers are inert to Li(+) ions? Identification of this novel member of the Na(+)/Ca(2+) superfamily, with distinct characteristics, including the ability to transport Li(+), may provide an explanation for this phenomenon.

Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii.

The effect of salinity on the antioxidative system of root mitochondria and peroxisomes of a cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) was studied. Salt stress induced oxidative stress in Lem mitochondria, as indicated by the increased levels of lipid peroxidation and H(2)O(2). These changes were associated with decreased activities of superoxide dismutase (SOD) and guaiacol peroxidases (POD) and contents of ascorbate (ASC) and glutathione (GSH). By contrast, in mitochondria of salt-treated Lpa plants both H(2)O(2) and lipid peroxidation levels decreased while the levels of ASC and GSH and activities of SOD, several isoforms of ascorbate peroxidase (APX), and POD increased. Similarly to mitochondria, peroxisomes isolated from roots of salt-treated Lpa plants exhibited also decreased levels of lipid peroxidation and H(2)O(2) and increased SOD, ascorbate peroxidase (APX), and catalase (CAT) activities. In spite of the fact that salt stress decreased activities of antioxidant enzymes in Lem peroxisome, oxidative stress was not evident in these organelles.

Coordinate induction of glutathione biosynthesis and glutathione-metabolizing enzymes is correlated with salt tolerance in tomato.

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that differ in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of gamma-glutamylcysteine synthetase protein, an effect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species.

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H2O2 level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.

Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: increased activities of antioxidant enzymes in root plastids.

Root plastids of the cultivated tomato Lycopersicon esculentum (Lem) exhibited salt-induced oxidative stress as indicated by the increased H2O2 and lipid peroxidation levels which were accompanied with increased contents of the oxidized forms of ascorbate and glutathione. In contrast, H2O2 level decreased, lipid peroxidation level slightly decreased and the levels of the reduced forms of ascorbate and glutathione increased in plastids of L. pennellii (Lpa) species in response to salinity. This better protection of Lpa root plastids from salt-induced oxidative stress was correlated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (POD), monodehydroascorbate reductase (MDHAR), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). In the plastids of both species, activities of SOD, APX, and POD could be resolved into several isozymes. In Lem plastids two Cu/ZnSOD isozymes were found whereas in Lpa an additional FeSOD type could also be detected. In response to salinity, activities of selected SOD, APX, and POD isozymes were increased in Lpa, while in Lem plastids the activities of most of SOD and POD isozymes decreased. Taken together, it is suggested that plastids play an important role in the adaptation of Lpa roots to salinity.

Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: The root antioxidative system.

The response of the antioxidant system to salt stress was studied in the roots of the cultivated tomato Lycopersicon esculentum Mill. cv. M82 (Lem) and its wild salt-tolerant relative L. pennellii (Corr.) D'Arcy accession Atico (Lpa). Roots of control and salt (100 mM NaCl)-stressed plants were sampled at various times after commencement of salinization. A gradual increase in the membrane lipid peroxidation in salt-stressed root of Lem was accompanied with decreased activities of the antioxidant enzymes: superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and decreased contents of the antioxidants ascorbate and glutathione and their redox states. In contrast, increased activities of the SOD, CAT, APX, monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), and increased contents of the reduced forms of ascorbate and glutathione and their redox states were found in salt-stressed roots of Lpa, in which the level of membrane lipid peroxidation remained unchanged. It seems that the better protection of Lpa roots from salt-induced oxidative damage results, at least partially, from the increased activity of their antioxidative system.