Defense Responses in Grapevine Leaves Against Botrytis cinerea Induced by Application of a Pythium oligandrum Strain or Its Elicitin, Oligandrin, to Roots.

ABSTRACT Pythium oligandrum is known to display antagonistic activities against several species of pathogenic fungi. It also produces an elicitor of plant defense named oligandrin, which belongs to the elicitin family (10-kDa proteins synthesized by Phytophthora and Pythium species). Here, the potential of P. oligandrum or its purified elicitin to limit the progression of B. cinerea on grapevine leaf and the resulting plant-microorganism interactions are described. P. oligandrum or oligandrin were applied to roots, and changes in the ultrastructure and at the molecular level were examined. When B. cinerea was applied to leaves of pretreated plants, leaf invasion was limited and the protection level reached about 75%. On leaf tissues surrounding B. cinerea inoculation, modifications of cuticle thickness, accumulation of phenolic compounds, and cell wall apposition were observed, indicating that grapevine can be considered reactive to elicitins. No macroscopic hypersensitive reaction associated with the elicitation treatment was observed. At the molecular level, the expression of three defense-related genes (LTP-1, beta-1,3-glucanase, and stilbene synthase) was studied. RNAs isolated from B. cinerea-infected leaves of grapevine challenged or not with P. oligandrum or oligandrin were analyzed by real-time reverse transcription-polymerase chain reaction. In grapevine leaves, LTP-1 gene expression was enhanced in response to oligandrin, and RNA transcript levels of beta-1,3-glucanase and stilbene synthase increased in response to all treatments with different magnitude. Taken together, these results open new discussion on the concept of plant reactivity to elicitins, which has until now, been mainly based on plant hypersensitive responses.

Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.

Computerized measurement of the three-dimensional distribution of optic disc pallor.

PURPOSE: To describe a method which provides quantitative measurements of the surface area of pallor in each quadrant of the three-dimensional optic cup, using photogrammetric measurements from simultaneous stereophotographs and computerized image analysis. METHODS: Simultaneous stereophotographs of one normal subject and two subjects with primary open angle glaucoma were digitized and analyzed for depth measurements. The boundaries of the optic disc, optic cup and region of pallor were identified. Pallor/disc and pallor/cup ratios were subsequently calculated for the superior, temporal, inferior and nasal walls. RESULTS: A digitized photograph and a Laplacian-filtered image were obtained for each eye to be studied. After processing each stereo pair through a similarity sequential detection-based algorithm, depth measurements are represented as a grey scale image, a contour plot, and a wire mesh, with the boundaries of the optic disc, optic cup and pallor superimposed. Ratios are given of the surface area of pallor to the surface area of the disc and the surface area of pallor to the surface area of the cup, by quadrant. CONCLUSIONS: Determination of surface area of pallor to cup may be useful in detecting early visual field loss in glaucoma and neurological disease.

Vehicle travel speeds and the incidence of fatal pedestrian crashes.

The aim of this study was to estimate the likely effect of reduced travel speeds on the incidence of pedestrian fatalities in Adelaide, Australia. The study was based on the results of detailed investigations of 176 fatal pedestrian crashes in the Adelaide area between 1983 and 1991. The method developed to estimate the effect of reduced travelling speed is described and supported by references to the published literature. A reduction in the speed limit from 60 to 50 km/h was one of four speed reduction scenarios considered. The smallest estimated reduction in fatal pedestrian collisions in the selection presented was 13%, for a scenario in which all drivers obeyed the existing speed limit. The largest estimated reduction was 48% for a scenario in which all drivers were travelling 10 km/h slower. The estimated reductions in fatalities obtained in this study are compared with those observed in places where the urban area speed limit has been lowered.

Human cysteine dioxygenase type I (CDO-I; EC 1.13.11.20): 5' flanking region and intron-exon structure of the gene.

AIM: The elucidation of the structure of the 5' flanking region and the exonic organisation of the human cysteine dioxygenase type I gene. METHODS: Material for sequence studies was generated by polymerase chain reaction (PCR) methods using human genomic DNA, a cosmid clone containing the entire gene, and a commercial gene walking kit. RESULTS AND CONCLUSIONS: The gene was found to be--12 kb in length and made up of five exons (418, 78, 155, 170, and 731 base pairs, respectively). The immediate 5' flanking region did not contain the canonical TATAA box. One DNA source (Clontech promoter finder kit) contained a liver specific nuclear factor response element approximately 550 base pairs from the transcription start site, whereas a second source did not. This and other cis acting factors in the 5' flanking region were identified by computer analysis. The physiological significance of such elements requires detailed experimental evaluation.

The structure of the human thyroxine binding globulin (TBG) gene.

A portion of the human X-chromosome (> 5 kb) encoding the translated portion of the thyroxine-binding globulin (TBG) gene was sequenced. The primary templates for sequencing were isolated from a human X-chromosome library (two positive plaques from 400,000 screened initially with a TBG cDNA probe) or were produced by PCR amplification using leucocyte genomic DNA as the amplification template. Potential hormone response elements (HREs) were identified at either end of the gene. These HREs have sequences based on the consensus half-site of thyroid hormone response elements, although it is unclear whether the structures are functional HREs. Other potential regulatory elements also were identified towards the 3' end of the gene.

Is oleic acid the thyroxine binding inhibitor in the serum of ill patients?

The possibility that oleic acid is the thyroxine binding inhibitor in the serum of seriously ill patients was investigated. 3H-Oleic acid was shown to bind directly to human thyroxine-binding globulin (TBG) by the techniques of one and two-dimensional immunoelectrophoresis in combination with autoradiography. However, no correlation was seen between serum thyroxine concentration and oleic acid concentration in two groups of patients, one of which underwent routine cholecystectomy, whilst the other group was admitted to an intensive therapy unit (mortality 75%). No correlation was seen between serum total thyroxine concentration and either stearic, palmitic, linoleic or arachidonic acid concentrations in these groups. Therefore, it was concluded that oleic acid was unlikely to be the circulating inhibitor of thyroxine binding.

Development of a gas chromatographic selected ion monitoring assay for thyroxine (T4) in human serum.

The components of a gas chromatographic mass spectrometric-selected ion monitoring (SIM) assay for thyroxine (T4) in human serum are described. The internal standard for the assay was synthesized from deuterium-labelled 3,5-diiodotyrosine and 3,5-diiodo-4-hydroxyphenylpyruvic acid. A novel method was developed for isolating the products of the coupling reaction. The results obtained by gas chromatography mass spectrometry SIM were compared with those of radioimmunoassay. The gas chromatographic mass spectrometric SIM assay would form the basis of a reference assay for T4.