Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavage-religation equilibrium, increasing single-strand nicks and protein-DNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.     

Mapping of AFLP markers linked to seed coat colour loci in<Emphasis Type="Italic"> Brassica juncea</Emphasis> (L.) Czern

Association mapping of the seed-coat colour with amplified fragment length polymorphism (AFLP) markers was carried out in 39 Brassica juncea lines. The lines had genetically diverse parentages and varied for seed-coat colour and other morphological characters. Eleven AFLP primer combinations were used to screen the 39 B. juncea lines, and a total of 335 polymorphic bands were detected. The bands were analysed for association with seed-coat colour using multiple regression analysis. This analysis revealed 15 markers associated with seed-coat colour, obtained with eight AFLP primer combinations. The marker E-ACA/M-CTG₃₅₀ explained 69% of the variation in seed-coat colour. This marker along with markers E-AAC/M-CTC₂₃₅ and E-AAC/M-CTA₂₅₀ explained 89% of the total variation. The 15 associated markers were validated for linkage with the seed-coat colour loci using a recombinant inbred line (RIL) mapping population. Bands were amplified with the eight AFLP primer combinations in 54 RIL progenies. Of the 15 associated markers, 11 mapped on two linkage groups. Eight markers were placed on linkage group 1 at a marker density of 6.0 cM, while the remaining three were mapped on linkage group 2 at a marker density of 3.6 cM. Marker E-ACA/M-CTG₃₅₀ co-segregated with Gene1 controlling seed-coat colour; it was specific for yellow seed-coat colour and mapped to linkage group 1. Marker E-AAC/M-CTC₂₃₅ (AFLP8), which had been studied previously, was present on linkage group 2; it was specific for brown seed-coat colour. Since AFLP markers are not adapted for large-scale applications in plant breeding, it is important to convert these to sequence-characterised amplified region (SCAR) markers. Marker E-AAC/M-CTC₂₃₅ (AFLP8) had been previously converted into a SCAR. Work is in progress to convert the second of the linked markers, E-ACA/M-CTG₃₅₀, to a SCAR. The two linked AFLP markers converted to SCARs will be useful for developing yellow-seeded B. juncea lines by means of marker-assisted selection.Communicated by H.F. Linskens     

Parasite-Antigen Driven Expansion of IL-5? and IL-5+ Th2 Human Subpopulations in Lymphatic Filariasis and Their Differential Dependence on IL-10 and TGFβ

Background

Two different Th2 subsets have been defined recently on the basis of IL-5 expression – an IL-5⁺Th2 subset and an IL-5⁻Th2 subset in the setting of allergy. However, the role of these newly described CD4⁺ T cells subpopulations has not been explored in other contexts.

Methods

To study the role of the Th2 subpopulation in a chronic, tissue invasive parasitic infection (lymphatic filariasis), we examined the frequency of IL-5⁺IL-4⁺IL-13⁺ CD4⁺ T cells and IL-5⁻IL-4 IL-13⁺ CD4⁺ T cells in asymptomatic, infected individuals (INF) and compared them to frequencies (F_o) in filarial-uninfected (UN) individuals and to those with filarial lymphedema (CP).

Results

INF individuals exhibited a significant increase in the spontaneously expressed and antigen-induced F_o of both Th2 subpopulations compared to the UN and CP. Interestingly, there was a positive correlation between the F_o of IL-5⁺Th2 cells and the absolute eosinophil and neutrophil counts; in addition there was a positive correlation between the frequency of the CD4⁺IL-5⁻Th2 subpopulation and the levels of parasite antigen – specific IgE and IgG4 in INF individuals. Moreover, blockade of IL-10 and/or TGFβ demonstrated that each of these 2 regulatory cytokines exert opposite effects on the different Th2 subsets. Finally, in those INF individuals cured of infection by anti-filarial therapy, there was a significantly decreased F_o of both Th2 subsets.

Conclusions

Our findings suggest that both IL-5⁺ and IL-5⁻Th2 cells play an important role in the regulation of immune responses in filarial infection and that these two Th2 subpopulations may be regulated by different cytokine-receptor mediated processes.     

In vitro activation of the IkappaB kinase complex by human T-cell leukemia virus type-1 Tax

Human T-cell leukemia virus type-I expresses Tax, a 40-kDa oncoprotein that activates IkappaB kinase (IKK), resulting in constitutive activation of NFkappaB. Herein, we have developed an in vitro signaling assay to analyze IKK complex activation by recombinant Tax. Using this assay in combination with reporter assays, we demonstrate that Tax-mediated activation of IKK is independent of phosphatases. We show that sustained activation of the Tax-mediated activation of the NFkappaB pathway is dependent on an intact Hsp90-IKK complex. By acetylating and thereby preventing activation of the IKK complex by the Yersinia effector YopJ, we demonstrate that Tax-mediated activation of the IKK complex requires a phosphorylation step. Our characterization of an in vitro signaling assay system for the mechanism of Tax-mediated activation of the IKK complex with a variety of mutants and inhibitors results in a working model for the biochemical mechanism of Tax-induced activation.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

In vitro regeneration of Psoralea corylifolia Linn.: influence of polyamines during in vitro shoot development

In Vitro Cellular & Developmental Biology - Plant - A reliable and economically feasible in vitro plant regeneration protocol has been standardized for the Psoralea corylifolia Linn. using...     

Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma

Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 T(H)1, T(H)2 and T(H)17 subsets. Interleukin-6 (IL-6) plays an important part in regulating these three arms of the immune response by limiting the T(H)1 response and promoting the T(H)2 and T(H)17 responses. In this study, we investigated pathways in DCs that promote IL-6 production. We show that the allergen house dust mite (HDM) or the mucosal adjuvant cholera toxin promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures established from the lung-draining lymph nodes of immunized wild-type mice blunted the T(H)2 and T(H)17 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or cholera toxin. DCs expressing nonfunctional c-Kit were unable to induce a robust T(H)2 or T(H)17 response and elicited diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand Jagged-2, which has been associated with T(H)2 differentiation, was blunted in DCs from c-Kit-mutant mice. c-Kit upregulation was specifically induced by T(H)2- and T(H)17-skewing stimuli, as the T(H)1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase (p110(D910A)) secreted lower amounts of IL-6 upon stimulation with cholera toxin. Collectively, these results highlight the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in regulating T cell responses.     

Cooperative activity of DNA methyltransferases for maintenance of symmetrical and non-symmetrical cytosine methylation in Arabidopsis thaliana

Maintenance of cytosine methylation in plants is controlled by three DNA methyltransferases. MET1 maintains CG methylation, and DRM1/2 and CMT3 act redundantly to enforce non-CG methylation. RPS, a repetitive hypermethylated DNA fragment from Petunia hybrida, attracts DNA methylation when transferred into Petunia or other species. In Arabidopsis thaliana, which does not contain any RPS homologues, RPS transgenes are efficiently methylated in all sequence contexts. To test which DNA methylation pathways regulate RPS methylation, we examined maintenance of RPS methylation in various mutant backgrounds. Surprisingly, CG methylation was lost in a drm1/2/cmt3 mutant, and non-CG methylation was almost completely eliminated in a met1 mutant. An unusual cooperative activity of all three DNA methyltransferases is therefore required for maintenance of both CG and non-CG methylation in RPS. Other unusual features of RPS methylation are the independence of its non-CG methylation from the RNA-directed DNA methylation (RdDM) pathway and the exceptional maintenance of methylation at a CC(m)TGG site in some epigenetic mutants. This is indicative of activity of a methylation system in plants that may have evolved from the DCM methylation system that controls CC(m)WGG methylation in bacteria. Our data suggest that strict separation of CG and non-CG methylation pathways does not apply to all target regions, and that caution is required in generalizing methylation data obtained for individual genomic regions.