‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Cyclooxygenase ‐2 Expression in Mammary Tumors in Dogs and its Correlation to Histologic and Biologic Behavior

Introduction:  Cyclooxygenase‐2 (Cox‐2) is the inducible form and the rate‐limiting enzyme, for conversion of arachidonic acid to prostaglandins. Cox‐2 overexpression, common in carcinomas, is associated with increased growth rate, resistance to apoptosis, angiogenesis, and overall, both local and distant aggressive behavior. Cox‐2 overexpression has been detected in human and canine mammary tumors (MTs). Histopathology of canine MT is not always predictive of biologic behavior, and anecdotally, only 50% of the malignant MTs are expected to metastasize. We hypothesize that Cox‐2 expression correlates with aggressive behavior.
Methods:  This retrospective study evaluated 48 bitches, presented for excision of MT between 2000 and 2003 at FMVZ de Botucatu‐UNESP, Brasil. Follow‐up varied from 18 months to 24 months and included physical examination and thoracic radiographs. Histopathologic examination was performed in all tumors, as well as in metastatic lesions when detected in the follow‐up period. Immunohistochemistry was used to detect expression of Cox‐2 in paraffin blocks (Rabbit polyclonal anti‐PGHS‐2. Oxford Biomedical). 10 adenomas, 10 carcinomas, 10 benign mixed tumors, 10 malignant mixed tumors and 8 cases of primary carcinomas and their metastatic lesions.
Results:  Expression of Cox‐2 varied among groups. Adenomas (32.1%), mixed benign tumors (38%), carcinomas (60.3%), malignant mixed tumors (65.8%), and metastatic carcinomas (81.25%) and their metastatic lesions (84.35%). Statistically significant differences (p < 0.05) were observed between the benign and malignant counterparts and between carcinomas and metastatic carcinomas.
Conclusions:  Cox‐2 expression correlates with both histologic and biologic behavior in mammary carcinomas, and may serve as a predictor of metastatic potential.     

Different biological behaviour of Waldenström macroglobulinemia in two dogs

Waldenström Macroglobulinemia is a low‐grade immunosecretory disorder associated with lymphoid tumours, which is rarely reported in veterinary medicine. In this study, we describe two clinical cases of this rare syndrome in dogs, each characterized by a different onset and clinical course. In one case, a hyperacute onset and aggressive behaviour of the neoplasm was observed. Absolute serum viscosity (SV) was retrospectively evaluated in order to explain clinical findings. Rotational viscosimetry showed good precision in measuring SV. Both dogs had SV values higher than a control groups of healthy dogs although only one subject developed hyperviscosity symptoms and complications. At high paraprotein concentrations, a slight reduction of the M‐component was associated with a marked decrease in SV. Thus, this work suggests that SV assessment is a relevant tool for managing monoclonal gammopathies, whose usefulness should be further confirmed in larger cohorts of dogs.     

剡溪山水文化旅游资源开发设想

文化底蕴越是丰厚的旅游资源，开发价值就越高。正是众多文人贤士游剡访古，使浙东剡溪的历史、文化与自然山水完美地结合在一起，形成了绚丽多彩的剡溪山水文化。文化被认为是旅游业的灵魂，是一种潜在的旅游产品。要提高山水旅游产品的文化品位，必须将山水文化的潜在价值转化为旅游产品。本文对剡溪山水文化旅游资源的开发提出了以下设想：再现“唐诗之路”山水旅游美景，并着重于按唐诗及有关文献、传说等增添新景点，沿江、沿山、于水中进行山水、文化全面开发，形成立体式、全景式的剡中山水文化游道；可开发出文化生态、修学、科考、宗教、节庆等剡溪山水文化旅游产品。     

论个性化的大学英语课程设置

讨论了目前大学英语课程设置单一的现状，并分析其形成原因。文章以ESP研究关于需求分析的理论为依据，提出大学应根据学生的实际需要、新时期国家和社会对人才培养需要，区别不同层次、不同程度、不同专业学生的需求，而专门设置一系列大学英语课程。说明了设置个性化的大学英语课程的理由和好处。     

Does matrix resistance influence Red squirrel (Sciurus vulgaris L. 1758) distribution in an urban landscape?

In determining isolation effects in fragmented populations, the landscape matrix is not often considered. Usually simple distance measures are used to quantify degree of isolation. We tested the effect of the matrix on the presence of red squirrels in 354 wooded patches in the Brussels Region, by comparing several isolation measures. These were 1) distance to the nearest source patch, 2) the Hanski-measure (a combination of distance to and size of all possible sources), 3) effective distances calculated from different least cost models using the ArcView grid extension ‘Cost Distance’ (a combination of distance and resistance of the landscape, with different resistances for different landscape types) and 4) some combinations of the Hanski-measure and the effective distances. Size and quality of the target patches were always included in the tests of the predictive power of different isolation measures on squirrel presence/absence. All variables examined (patch size, quality and isolation) significantly influenced squirrel presence. Models using the effective distances gave the best results. Models including the Hanski-measure improved significantly when Euclidean distance was replaced by effective distance, showing that parameterisation of matrix resistance added significant additional explanatory power when modelling squirrel presence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Secretion of Natural and Synthetic Toxic Compounds from Filamentous Fungi by Membrane Transporters of the ATP-binding Cassette and Major Facilitator Superfamily

This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environment of fungi, such as antibiotics produced by other microorganisms. In plant pathogenic fungi, these transporters can also be an important determinant of virulence on host plants by providing protection against plant defence compounds or mediating the secretion of host-specific toxins. Furthermore, they play a critical role in determining base-line sensitivity to fungicides and other antimycotic agents. Overexpression of some of these transporters can lead to the development of resistance to chemically-unrelated compounds, a phenomenon described as multidrug resistance (MDR). This has been observed in a variety of organisms and can impose a serious threat to the effective control of pathogenic fungi.     

The pupal morphology of Cameraria ohridella compared with that of the genus Phyllonorycter (Lepidoptera: Gracillariidae)

The pupal morphology of the pest species Cameraria ohridella is described and compared with that of the genus Phyllonorycter. The most striking differences are the longer antennal appendages, the absence of a cremaster, the presence of strong lateral thorns on abdominal segments 1–5 and an area without small spines on the same abdominal segments in Cameraria.