The gram negative, microaerophilic bacterium Helicobacter I~Iori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. ~lori pathogenesis and the development of therapeutics and vaccines. The recently discovered, gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma (1-5). H. pylori infection is a worldwide problem, since in developing countries it affects over 80% of the population older than 20. Also in developed countries the infection is present in 20% of the population by the age of 30 rising to over 50% by the age of 60. Clinical isolates of H. pylori can be classified into two groups based on the presence or absence of the vacuolating cytotoxin (6, 7) whose expression is linked to a surface exposed immunodominant antigen (CagA) (8, 9). Since high titers of serum antibodies to the CagA protein are detected in all patients with duodenal ulcer (8) and most of those with gastric carcinoma (10, 11), it has been proposed that disease development requires infection with cytotoxin-producing strains.The cytotoxin causes massive vacuolation in several mammalian cell lines (6), and similar vacuoles have also been observed in the gastric epithelia of patients with active chronic gastritis associated with H. pylori infection (12), indicating that the cytotoxin can contribute significantly to the pathogenesis of gastritis. Cell vacuolation in vitro can be blocked and reversed by inhibitors of V-type ATPases and potentiated by inhibitors of the Na+-K + ATPase (13,14), suggesting that the mechanism of action of the toxin is due to aberrant cation transport within the target cells. The purified toxin has been described as a protein of ~87 kD that is found in the bacterial culture supernatants, and the sequence of the NH2-terminal 23 amino acids has been determined (7).Despite the epidemiological correlation between infection with cytotoxic strains and disease (8) and the in vitro evidence for the presence of a cytotoxin, the in vivo roles of infection and cytotoxin have not been established due to the lack of a suitable animal model. H. ~lori does not colonize the gastric mucosa of mice or other small laboratory animals.To overcome this limitation, we administered H. pylori extracts and purified cytotoxin orally to mice. Using this model, extracts from cytotoxic H. Ioylori strains and purified cytotoxin ind...

Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.

Pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. We selected a single-chain antibody fragment (scFvLR3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (GLRaV-3), one of the agents of grapevine leafroll (GLR) disease, from a phage display library. The antibody binds specifically to the entire length of GLRaV-3 particles and has a high binding affinity value (K(D)) of 42 nM. The amino acid motif AQEPPRQ located at the N terminus of the GLRaV-3 coat protein was identified as the antibody-binding epitope by PEPSCAN analysis. To evaluate scFv stability in the reducing environment of the plant cell cytosol, transient expression assays were performed using Nicotiana benthamiana as a model plant. Capture ELISA demonstrated that the scFv fragment was produced and retained its antigen-binding capacity in the plant cytosol. Further functional assays showed that scFvLR3cp-1 binds with high specificity to at least four members of the family Closteroviridae. Therefore, the GLRaV-3-specific scFv fragment could be an ideal candidate for mediating broad-spectrum virus resistance if produced in transgenic grapevine plants.

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