A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I beta-1,3-glucanase against Fusarium solani germlings.     

Optimality-criteria-based probabilistic structural design

The possibility of extending absolute minimum weight designs for sandwich beam systems or fibre-reinforced beams and plates is considered when both the extreme loading applied to the system and the beam bending resistance properties are described in probabilistic rather than deterministic terms. It is shown that such an extension is possible provided certain plausible assumptions are made about the probabilistic modelling of the system. Examples considered include simple and continuous beams under loading of uncertain magnitude, direction or location. It is shown that the classical (deterministic) optimal solutions remain valid under reasonable assumptions. The extension of the probabilistic solutions to a wider class of examples is discussed. Received October 20, 2000     

Nonlinear wave theory in reliability analysis of offshore structures

The reliability analysis of offshore structures under wave and wind actions is considered using second order random wave theory. To represent non-Gaussian properties of the resulting wave kinematics, the Hermite moment transformation is used. Further, the so-called sample-specific linearization method developed already (to be used in conjunction with the directional simulation method and the linear wave theory) will be extended to take into account both (1) non-Gaussianity of wave/wind load due to nonlinear load processes and also (2) the non-Gaussianity of wave kinematics due to the nonlinear wave theory. This allows an out-crossing approach to be used to assess the structural probability of failure and the involving out-crossing rate (which is not generally available for non-Gaussian processes) is required to be estimated. Using the proposed procedure, simple structures are analyzed in one- and multi-dimensional cases and the results for structural probability of failure are compared with those obtained using simple linear wave theory. Outcomes show that the use of nonlinear wave theory may affect the results considerably.     

Load combination analysis by ‘Directional simulation in the load space’

The reliability of structures subjected to multiple time-varying random loads is considered herein. It is well-known that the reliability of such systems may be evaluated by considering outcrossings of the load process vector out of a safe domain, and the contribution of individual loads to structural failure may be evaluated by considering outcrossings caused by combinations of one or more loads. In this paper the ‘Directional Simulation in the Load Space’ approach to reliability analysis is developed to consider explicitly outcrossings caused by all possible combinations of loads, during analysis of systems comprising stationary continuous Gaussian loads. To do this, the direction of the load process vector is ‘fixed’ at each point of outcrossing (to physically represent the particular combination of loads causing the outcrossing), and, by considering each possible load combination, all loads not causing an outcrossing are then held constant during radial integration (to model correctly that they do not contribute to each outcrossing). A numerical example demonstrating the validity of the proposed formulation is presented.     

Corrosion and capacity prediction of marine steel infrastructure under a changing environment

The deterioration of marine steel infrastructure caused by corrosion may be influenced by a changing climate and/or pollution level which may lead to its serviceability and structural failure. However, almost all corrosion research until recently assumed time-invariant environmental conditions. A structural reliability analysis is applied here to simulate steel sheet piles in sea water conditions under a changing environment. Corrosion of marine steel infrastructure is modelled as a spatial time-dependent process including sea water temperature and sea level rise due to global warming and dissolved inorganic nitrogen concentration increase caused by pollution. The steel sheet piles are divided vertically into 70 elements to consider the spatial variability of different corrosion zones and sea level rise effects. Two limit states are considered: (i) stress of steel sheet piles reaches their yield stress and (ii) pitting corrosion perforation to provide an alert to repair or maintenance. The results show that ignoring the effects of a changing environment can underestimate structural capacity failure risks, and pollution will have a more significant effect on capacity of steel sheet piles than sea water temperature and sea level rise caused by global warming.     

Reinforcement corrosion initiation and activation times in concrete structures exposed to severe marine environments

The corrosion of steel reinforcement bars in reinforced concrete structures exposed to severe marine environments usually is attributed to the aggressive nature of chloride ions. In some cases in practice corrosion has been observed to commence already within a few years of exposure even with considerable concrete cover to the reinforcement and apparently high quality concretes. However, there are a number of other cases in practice for which corrosion initiation took much longer, even in cases with quite modest concrete cover and modest concrete quality. Many of these structures show satisfactory long-term structural performance, despite having high levels of localized chloride concentrations at the reinforcement. This disparity was noted already more than 50 years ago, but appears still not fully explained. This paper presents a systematic overview of cases reported in the engineering and corrosion literature and considers possible reasons for these differences. Consistent with observations by others, the data show that concretes made from blast furnace cements have better corrosion durability properties. The data also strongly suggest that concretes made with limestone or non-reactive dolomite aggregates or sufficiently high levels of other forms of calcium carbonates have favourable reinforcement corrosion properties. Both corrosion initiation and the onset of significant damage are delayed. Some possible reasons for this are explored briefly.     

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.     

Enzymatic amplification of lactate dehydrogenase-elevating virus

To improve the detection of lactate dehydrogenase-elevating virus (LDV), we developed a PCR assay. Primers were selected from ORF7, encoding nucleocapsid protein VP1. No specific amplification was observed with any other common murine virus or with RNAs from the closely related Lelystad virus and equine arteritis virus. In experimentally infected mice, LDV could be detected in plasma in both the acute and the persistent phases. LDV was also detected by the PCR in contaminated pools of Plasmodium berghei parasites which were maintained in mice, both by a direct analysis of the samples and by testing of plasma from mice inoculated with these pools. There was a complete agreement between the results of the PCR assay and the lactate dehydrogenase (LDH) enzyme assay of plasma from the inoculated mice. In contrast to the results of the LDH enzyme assay, no false-positive reactions were obtained in the PCR assay with negative control samples showing visible hemolysis. Storage of plasma samples at room temperature and at 4, -20, and -80 degrees C for up to 8 days did not influence the results of the PCR. These results show that the PCR is a valuable technique which may replace the LDH test as a diagnostic tool.