Oxidant-induced damage to the intima of pulmonary and systemic vessels is thought to be an important mechanism of injury in a variety of syndromes of vascular damage. Hydrogen peroxide (H202) is an active oxygen metabolite that may induce intimal injury by cytolytic attack or by inducing biochemical and functional alterations in the endothelial cells (EC); however, mechanisms involved in noncytolytic perturbation of EC are largely unknown. We found that H202 stimulated the synthesis of platelet-activating factor (PAF) by primary cultures of bovine pulmonary artery endothelium (BPAEC) and by human umbilical vein endothelium (HUVEC). In each cell type the incorporation of I3Hlacetate into 13H-acetylIPAF was concentration-and time-dependent and was temporally dissociated from severe plasma membrane disruption and cytolytic cell injury; the newly synthesized PAF remained associated with the EC. H202 caused permeabilization of EC to 45Ca2+ and an increase in intracellular Ca2+, suggesting that a transmembrane Ca2+ flux is the signal that initiates PAF synthesis.H202 also induced the endothelial cell-dependent adhesion of neutrophils to HUVEC monolayers. This response was rapid, with an onset within minutes and a subsequent time course that paralleled the time course of PAF accumulation, and was dependent on extracellular Ca2' but not on de novo protein synthesis. These studies demonstrate that H202 can induce two rapid activation responses of endothelium, PAF synthesis and EC-dependent neutrophil adhesion, events that may be important in physiologic and pathologic inflammation.

Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14-12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.

With established ampelographic techniques for grapevine identification it is often difficult to achieve a satisfactory, objective result. We have developed a DNA typing system using sequence-tagged microsatellite site markers as a means of differentiating cultivars of grapevine. A semi-automated analysis procedure was linked to an electronic database and found to be an objective and reliable system for cultivar identification using this simple marker type. The accumulated DNA typing data from over eighty cultivars demonstrated that cultivars that are difficult to differentiate phenotypically using ampelographic techniques can be distinguished by DNA typing. Parentage analysis uncovered errors in parent assignment or cultivar identification in specific cases. The electronic database has a conservative format to take into account the occurrence of null alleles and the possibility of missed alleles. Computer-assisted comparisons of cultivars in the database can be performed and various approaches for estimating the match probability that two unrelated cultivars have the same genotype simply due to chance are discussed. We suggest that further development of the database through international co-operation using standardised sequence-tagged site markers offers the possibility of achieving a universal grapevine identification system.

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