Gene silencing studies in the gymnosperm species Pinus radiata

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.     

Cell-free selection of zinc finger DNA-binding proteins using in vitro compartmentalization

We have exploited emulsion-based in vitro compartmentalization (IVC) to devise a method for the selection of zinc finger proteins (ZFPs) on the basis of their DNA-binding specificity. A library of ZFPs fused to a C-terminal peptide tag is encoded by a set of DNA cassettes that are prepared wholly in vitro. In addition to the ZFP gene, each DNA cassette also carries a given DNA target binding site sequence for which one wishes to isolate ZFP binders. An aliquot of the library is added to bacterial S30 extract and emulsified in mineral oil so that most of the aqueous droplets contain, on average, no more than one gene. If an intra-compartmentally expressed ZFP binds specifically to its encoding DNA via the target binding site, the complex can be purified by affinity capture via the peptide tag after breaking the emulsion, thus rescuing the gene. We present proof-of-principle for this IVC selection method by selecting a specific high-affinity ZFP gene from a high background of a related gene. We also propose that high-affinity ZFPs can be used as genotype-phenotype linkages to enable selection of other proteins using IVC.     

Pharmacological targeting of anaphylatoxin receptors during the effector phase of allergic asthma suppresses airway hyperresponsiveness and airway inflammation

Airway hyperresponsiveness and airway inflammation are hallmarks of allergic asthma, the etiology of which is crucially linked to the presence of Th2 cytokines. A role for the complement anaphylatoxins C3a and C5a in allergic asthma was suggested, as deficiencies of the C3a receptor (C3aR) and of complement factor C5 modulate airway hyperresponsiveness, airway inflammation, and Th2 cytokine levels. However, such models do not allow differentiation of effects on the sensitization phase and the effector phase of the allergic response, respectively. In this study, we determined the role of the anaphylatoxins on the effector phase of asthma by pharmacological targeting of the anaphylatoxin receptors. C3aR and C5a receptor (C5aR) signaling was blocked using the nonpeptidic C3aR antagonist SB290157 and the neutralizing C5aR mAb 20/70 in a murine model of Aspergillus fumigatus extract induced pulmonary allergy. Airway hyperresponsiveness was substantially improved after C5aR blockade but not after C3aR blockade. Airway inflammation was significantly reduced in mice treated with the C3aR antagonist or the anti-C5aR mAb, as demonstrated by reduced numbers of neutrophils and eosinophils in bronchoalveolar lavage fluid. Of note, C5aR but not C3aR inhibition reduced lymphocyte numbers in bronchoalveolar lavage fluid. Cytokine levels of IL-5 and IL-13 in bronchoalveolar lavage fluid were not altered by C3aR or C5aR blockade. However, blockade of both anaphylatoxin receptors markedly reduced IL-4 levels. These data suggest an important and exclusive role for C5aR signaling on the development of airway hyperresponsiveness during pulmonary allergen challenge, whereas both anaphylatoxins contribute to airway inflammation and IL-4 production.     

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.     

Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans

Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates, alcohols and related compounds. Furthermore, the organism is used for several biotechnological processes, such as vitamin C production. To further our understanding of its overall metabolism, we sequenced the complete genome of G. oxydans 621H. The chromosome consists of 2,702,173 base pairs and contains 2,432 open reading frames. In addition, five plasmids were identified that comprised 232 open reading frames. The sequence data can be used for metabolic reconstruction of the pathways leading to industrially important products derived from sugars and alcohols. Although the respiratory chain of G. oxydans was found to be rather simple, the organism contains many membrane-bound dehydrogenases that are critical for the incomplete oxidation of biotechnologically important substrates. Moreover, the genome project revealed the unique biochemistry of G. oxydans with respect to the process of incomplete oxidation.     

Prokaryotic sulfur oxidation

Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the beta and gamma subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiotic bacteria.     

Invasive versus noninvasive measurement of allergic and cholinergic airway responsiveness in mice

Background

This study seeks to compare the ability of repeatable invasive and noninvasive lung function methods to assess allergen-specific and cholinergic airway responsiveness (AR) in intact, spontaneously breathing BALB/c mice.

Methods

Using noninvasive head-out body plethysmography and the decrease in tidal midexpiratory flow (EF₅₀), we determined early AR (EAR) to inhaled Aspergillus fumigatus antigens in conscious mice. These measurements were paralleled by invasive determination of pulmonary conductance (GL), dynamic compliance (Cdyn) and EF₅₀ in another group of anesthetized, orotracheally intubated mice.

Results

With both methods, allergic mice, sensitized and boosted with A. fumigatus, elicited allergen-specific EAR to A. fumigatus (p < 0.05 versus controls). Dose-response studies to aerosolized methacholine (MCh) were performed in the same animals 48 h later, showing that allergic mice relative to controls were distinctly more responsive (p < 0.05) and revealed acute airway inflammation as evidenced from increased eosinophils and lymphocytes in bronchoalveolar lavage.

Conclusion

We conclude that invasive and noninvasive pulmonary function tests are capable of detecting both allergen-specific and cholinergic AR in intact, allergic mice. The invasive determination of GL and Cdyn is superior in sensitivity, whereas the noninvasive EF₅₀ method is particularly appropriate for quick and repeatable screening of respiratory function in large numbers of conscious mice.     

A study of porous structure of cellular membranes in human erythrocytes

Properties of cell membrane of human erythrocytes are studied using the mechanistic formalism of membrane transport developed earlier. We estimate that an erythrocyte with a membrane surface of 176 x 10(6)nm2 has about 1900 water-permeable pores with cross-section areas ranging from 0.07 to 0.2 nm2.     

Summary of the animal homologue section of HLDA8

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round of testing by flow cytometry (FCM) 182 mAb showed reactivity with atleast one of the species described above. Most of the cross-reactivity was found against non-human primate leukocytes, but also species in evolutionarily more distant from humans showed in some cases a clear staining pattern in flow cytometry (FCM). In a second round these FCM-results were confirmed by molecular analyses, by immunoprecipitation studies and analyses on transfectants. Interesting was the broad species-overlapping reactivity of mAb directed against CD9 (11 out of 17 species), CD11a (11/17), CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), and CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of crossreactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.     

SPOT synthesis: reliability of array-based measurement of peptide binding affinity

Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing peptide arrays for protein binding really reflect the dissociation constants of the protein-peptide complexes. Using the monoclonal antibody CB4-1 as a model system, we systematically compared dissociation constants of antibody-peptide complexes with signal intensities obtained using the SPOT technology. By analyzing a set of peptides possessing different affinities to the antibody, we determined the strengths of the SPOT screening method. The accuracy of the measured results was improved by taking regional trends in the membrane surface into account. A model based on the mass action law compares well with the experimental results. Interestingly, the applied concentrations of the binding partners do not directly correspond to the effective concentrations in the assay. We show that the SPOT technology is an accurate method for assigning the spots' measured signal intensities to three different binding affinity classes. The dissociation constants of the intermediate region were found to be between pK(dis)=5 and pK(dis)=7. Altering the experimental parameters causes a directed change of this region.