Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.     

Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity

Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.     

Gravitropism in Phycomyces: threshold determination on a clinostat centrifuge

The absolute sensitivity of sporangiophores of Phycomyces blakesleeanus to centrifugal acceleration was determined on a clinostat centrifuge. The centrifuge provides centrifugal accelerations ranging from 10(-4) to 6 x g. The rotor of the centrifuge, which accommodates 96 culture vials with single sporangiophores, is clinostatted, that is, turning "head over", at slow speed (1 rev min(-1)) while it is running. The negative gravitropism of sporangiophores is characterized by two components: a polar angle, which is measured in the plane of bending, and an aiming-error angle, which indicates the deviation of the plane of bending from the vector of the centrifugal acceleration. Dose-response curves were generated for both angles with centrifugations lasting 3, 5, and 8 h. The threshold for the polar angle depends on the presence of statoliths, so-called octahedral protein crystals in the vacuoles. The albino strain C171 carAcarR (with crystals) has a threshold near 10(-2) x g while the albino strain C2 carAgeo-3 (without crystals) has a threshold of about 2 x 10(-1) x g. The threshold for the aiming error angle is ill defined and is between 10(-2) and 10(-1) x g. The threshold for the polar angle of the wild type NRRL 1555 (with crystals) is near 8 x 10(-2) x g.     

Cre recombinase-mediated gene targeting of mesenchymal cells

Loss-of-function approaches by the Cre/loxP technology have provided powerful tools for functional analyses of genes of interest expressed preferentially in a particular tissue. Here we describe the generation of transgenic mouse lines expressing Cre recombinase under the control of the promoter/enhancer unit of the gene for the alpha2 chain of collagen type I (Col1alpha2). As an expression vector, we used a P1-derived artificial chromosome (PAC), which harbors approximately 100 kb carrying the col1alpha2 gene. The improved coding sequence of the Cre recombinase was introduced to replace the first exon of col1alpha2. Cre expression was determined by immunohistochemistry and Cre-mediated onset of beta-galactosidase expression in ROSA26R-Cre reporter mice. In four analyzed transgenic lines, Cre recombinase was efficiently expressed during embryogenesis and in adult animals in cells of mesenchymal origin, such as dermal fibroblasts, mesenchymal cells of blood vessel walls, and cells in fibrous connective tissues surrounding internal organs.     

Fibroblast growth factors in epithelial repair and cytoprotection

Growth factors are polypeptides that stimulate the division of certain cell types at low concentrations. Fibroblast growth factor (FGF) 7 (FGF-7) and its homologue FGF-10 act specifically on various types of epithelial cells including keratinocytes of the skin, intestinal epithelial cells and hepatocytes. In addition, FGF-7 and FGF-10 have been shown to be more than growth factors: they can protect epithelial cells from damaging effects induced, for example, by radiation and oxidative stress. Therefore, they are currently in clinical trials for the treatment of oral mucositis, a severe side-effect of cancer therapy characterized by painful inflammation and ulceration of the oral epithelium. To gain insight into the mechanisms of FGF-7/FGF-10 action in epithelial cells, we searched for genes that are regulated by these growth factors. Indeed, we identified genes that help us to explain the mechanisms that underlie the effects of FGF-7. Most interestingly, several genes were identified that are likely to mediate the cytoprotective effect of FGF-7 for epithelial cells in vitro and possibly also in injured and diseased tissues in vivo.     

Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors

Local excess of nitric oxide (NO) has been implicated in beta-cell damage, thus, a possible approach to the treatment of autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahydropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently evaluated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells and a combination of IL1-1 beta and IFN-gamma as inducers of cellular NO production. The structure-activity analysis revealed that the variation of substituents in the position 1 of the hexahydropyridazine strongly influences the inhibitory activity to iNOS as well as being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly significant inhibitory effects. However, for an efficient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxyphenyl, and 4-methoxyphenyl (IC(50): 0.5-2.1 mM), respectively, provided compounds with similar inhibitory activity to aminoguanidine (IC(50): 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides, which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously substituted aromatic rings.