Assessing the performance of pea and lentil at different seeding densities as trap crops for the management of wireworms in spring wheat

Wireworms, the larvae of click beetles (Elateridae), are difficult to manage due to their habitats and behaviour. Wireworms pose a major threat to the wheat crop in the north‐western USA. Seed treatment with neonicotinoids, biological control management and some cultural controls are recommended to manage these pests. Trap cropping is an emerging way to manage wireworms. In strawberry and potato crops, trap cropping has been found effective at attracting wireworms away from the principal crop. An earlier study in the Golden Triangle area of Montana found that pea and lentil could be effective trap crops for managing wireworms in spring wheat. In the present study, experiments were conducted at two locations. The effectiveness of peas and lentils as trap crops with wheat at different seeding densities was assessed [pea at 0, 4, 8, 16 seeds/sq.ft. or 0, 43, 86, 172 seeds/sq.m.; lentil at 0, 6, 12, 18 OR 0, 65, 130, 194 seeds/sq.m.; both with wheat at 0, 11, 22, or 28 seeds/sq.ft. or 0, 120, 230, 300 seeds/sq.m.] in a randomized design where all three crops were intercropped. Both trap crops were found to be effective in protecting wheat at standard seeding rates of 8 seeds/sq.ft. for pea and 12 seeds/sq.ft. for lentils. At these seeding rates, higher numbers of wireworms were found to be attracted to the trap crop, resulting in higher yield (7%–10%) of the associated spring wheat plant stands at 22 seeds/sq.ft. To develop an effective trap crop strategy, the pea–wheat and lentil–wheat spatial patterns that are possible need to be assessed in further field trials. Proper design and evaluation of the cost–benefit ratio of pea and lentil as trap crops are likely to produce good results for wheat crops in Montana.     

Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.     

HupB,a Nucleoid-Associated Protein of Mycobacterium tuberculosis,Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H₃₇Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr⁶⁵ and Thr⁷⁴ in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg⁵⁵ is also identified as an important residue for N-HupB–DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H₃₇Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.     

Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.     

Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung

Background

The variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB.

Methods/Principal Findings

A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log₁₀, p<0.01) and spleen (2.36 log₁₀, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture.

Conclusions/Significance

These results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.