The ability of bacterial DNA methyltransferases to use methylcobalamine as a cofactor in DNA methylation reactions

The ability of bacterial DNA methyltransferases Alu I, Cfr I, Cfr 6, Cfr 10, Eco RI, Eco RII, Msp I, Mva I, Pvu I, Pvu II, and Sau 3A to use methylcobalamine and methylmethionine as cofactors of DNA methylation in vitro has been investigated. These bacterial DNA methyltransferases used methylcobalamine, but not methylmethionine for DNA methylation.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.     

Stimulated saprotrophic fungi in arable soil extend their activity to the rhizosphere and root microbiomes of crop seedlings

Saprotrophic fungi play an important role in ecosystem functioning and plant performance, but their abundance in intensively managed arable soils is low. Saprotrophic fungal biomass in arable soils can be enhanced with amendments of cellulose-rich materials. Here, we examined if sawdust-stimulated saprotrophic fungi extend their activity to the rhizosphere of crop seedlings and influence the composition and activity of other rhizosphere and root inhabitants. After growing carrot seedlings in sawdust-amended arable soil, we determined fungal and bacterial biomass and community structure in roots, rhizosphere and soil. Utilization of root exudates was assessed by stable isotope probing (SIP) following ¹³CO₂-pulse-labelling of seedlings. This was combined with analysis of lipid fatty acids (PLFA/NLFA-SIP) and nucleic acids (DNA-SIP). Sawdust-stimulated Sordariomycetes colonized the seedling's rhizosphere and roots and actively consumed root exudates. This did not reduce the abundance and activity of bacteria, yet higher proportions of α-Proteobacteria and Bacteroidia were seen. Biomass and activity of mycorrhizal fungi increased with sawdust amendments, whereas exudate consumption and root colonization by functional groups containing plant pathogens did not change. Sawdust amendment of arable soil enhanced abundance and exudate-consuming activity of saprotrophic fungi in the rhizosphere of crop seedlings and promoted potential beneficial microbial groups in root-associated microbiomes.     

Plectosphaerella cucumeria-occurrences as a new root rot pathogen and p-solubiliser in north-eastern India

Musa paradisiaca is an important fruit crop of tropical and subtropical countries in the world. In a survey conducted during 2009-2011, a new root rot pathogen was observed in more than 15% banana plants growing in the fields at Mizoram, India. Causative agent of the disease was identified as Plectosphaerella cucumeria and pathogenicity was determined by inoculating to healthy seedlings. This is the first report of the P.cucumeria as both new root rot pathogen and p-solubiliser.     

WISP-3 functions as a ligand and promotes superoxide dismutase activity

WISP-3 (Wnt1 inducible secreted protein-3) mutations have been linked to the connective tissue diseases progressive pseudorheumatoid dysplasia and polyarticular juvenile idiopathic arthritis, both of which are accompanied by disorders in cartilage maintenance/homeostasis. The molecular mechanism of WISP-3 mediated effects in the sustenance of cartilage has not been described in detail. Our previous study illustrates the potential role of WISP-3 in regulating the expression of cartilage-specific molecules that sustain chondrocyte growth and cartilage integrity. The present study was conducted to investigate the mode of action of WISP-3 in greater detail. Experimental results depicted here suggest that WISP-3 can function as a ligand and signal via autocrine and/or paracrine modes upon being secreted by chondrocytes. Furthermore, apart from regulating collagen II and aggrecan expression, WISP-3 may also promote superoxide dismutase expression and activity in chondrocytes.     

An artificial miRNA as a new tool to silence and explore gene functions in apple

Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar ‘Gala’. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

     

The ability of Vibrio vulnificus to use a synthetic hydrophilic heme compound, Fe-TPPS, as a single iron source

Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins. This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX). In the present study, we tested whether this pathogen also uses a synthetic heme compound, Fe-alpha,beta,gamma,delta-tetraphenylporphine tetrasulfonic acid (Fe-TPPS), as an iron source. When inoculated into a medium containing Fe-TPPS, V. vulnificus L-180 multiplication was seen to be dependent on the concentration of the synthetic heme compound; a mutant lacking the ability to utilize protoheme did not multiply. Cells of the strain grown under the iron-restricted condition showed time-dependent uptake of Fe-TPPS. The ability to use either protoheme or Fe-TPPS was significantly reduced by the addition of an excess amount of free TPPS or Cu-TPPS. The data suggest that, V. vulnificus may assimilate Fe-TPPS, at least partially, through the same system as that for protoheme.     

DFT study of new bipyrazole derivatives and their potential activity as corrosion inhibitors

In the present work, a theoretical study of five bipyrazolic-type organic compounds, 4-{bis[(3,5-dimethyl-1H-pyrazolyl-1-yl)methyl]-amino}phenol (1), N1,N1-bis[(3,5-dimethyl-1H-pyrazol-1-yl)methyl}]-N4,N4-dimethyl-1,4-benzenediamine (2), N,N-bis[(3,5-dimethyl-1H-pyrazol-1-yl)methyl]aniline (3), 4-[bis(3,5-dimethyl pyrazol-1-yl-methyl)-amino]butan-1-ol (4) and ethyl4-[bis(3,5-dimethyl-1H-pyrazol-1-yl-methyl) aminobenzoate] (5), has been performed using density functional theory (DFT) at the B3LYP/6-31G(d) level in order to elucidate the different inhibition efficiencies and reactive sites of these compounds as corrosion inhibitors. The efficiencies of corrosion inhibitors and the global chemical reactivity relate to some parameters, such as EHOMO, ELUMO, gap energy (ΔE) and other parameters, including electronegativity (χ), global hardness (η) and the fraction of electrons transferred from the inhibitor molecule to the metallic atom (ΔN). The calculated results are in agreement with the experimental data on the whole. In addition, the local reactivity has been analyzed through the Fukui function and condensed softness indices.     

Rodlet cells in teleosts: a new insight into their nature and functions

The nature of rodlet cells (RCs) and their functions is subject to a number of different interpretations. This review provides a detailed analysis of the parasitic and endogenous origin of these cells. Two new functional aspects of RCs are considered in detail. The possible function of RCs as immune cells was derived from studies that reported an increase in the number of RCs in fish infected with protozoan and metazoan parasites, particularly at the site of the pathogen infection and/or attachment. Accordingly, RCs represent inflammatory cells, with a similar role to eosinophile granule cells, epithelioid cells and mesothelial cells. Rodlet cells may potentially act as biomarkers. Experimental studies that examined the response of RCs in fish exposed to chemical substances such as metals and herbicides reported an increase in the number of RCs in the tissues of the fish. Fish exposed to these substances expressed myelinic figures in the cytoplasm of the RCs and various degrees of rodlet degeneration and high vacuolization of RC cytoplasm were often noticed. Further lines of research are suggested that might elucidate the true function of these enigmatic cells.     

The use of taurine analogues to investigate taurine functions and their potential therapeutic applications

Summary.  Despite the multitude of evidence for the beneficial effects of taurine supplementation in a variety of disease, the underlying modifying action of taurine with respect to either molecular or biochemical mechanisms is almost totally unknown. We have assessed the development of taurine analogues, particularly where there has been substitution at the suphonate or amine group. Such substitutions allow the investigator to probe the relationship between structure and function of the taurine molecule. In addition such studies should help to ascertain taurine's point of interaction with the effector molecule. These results will prepare the way for the development of the second generation of taurine analogues. Received January 2, 2002 Accepted January 28, 2002 Published online August 30, 2002 Acknowledgements This research has been funded by the COST Chemistry programmes COST D8 “Chemistry of Metals in Medicine” and D-13 “New Molecules for Human Health Care”. All of the authors are members of the Working Group D13/0011/00 “Investigation of mechanisms underlying the pharmacological actions of taurine upon cell apoptosis and calcium homeostasis”. Authors' address: Dr. R.J. Ward, Unite de Biochimie, Catholic Universite de Louvain, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium, E-mail: ward@bioc.ucl.ac.be     

Transgenic foods as a tool for malnutrition elimination and their impact on agricultural systems.

GMO crops were introduced for commercial production in 1996. Since then, their use has increased rapidly. GMOs have primarily benefited large farms and multinational companies in Industrialised Countries and now is more and more debating their utilisation in Developing World. The objective of the present review is an analysis of this subject from a comprehensive point of view; in addition to that, the changes related to the nutritional content of transgenic foods will be treated. Despite the progress that has been made, the world food situation is still marked by mass hunger and chronic malnutrition. In particular micronutrient malnutrition, that means vitamin and mineral deficiencies, represents an important public health problem in several areas of the world. The "golden rice" bioengineered to contain beta-carotene, as a source of vitamin A is the most famous example of GM food used for reduction (or even to solve) of a public health problem. The expected results of this approach have presently not been achieved. Further studies are necessary to increase the general knowledge about GMOs and their long-term effects on human health. Collaborative attitude of different research sectors (private and public) and involvement of different sectors of society will be an added value for comprehension of the real impact of the application of modern biotechnology to food and agriculture systems.     

Invasive ants carry novel viruses in their new range and form reservoirs for a honeybee pathogen

When exotic animal species invade new environments they also bring an often unknown microbial diversity, including pathogens. We describe a novel and widely distributed virus in one of the most globally widespread, abundant and damaging invasive ants (Argentine ants, Linepithema humile). The Linepithema humile virus 1 is a dicistrovirus, a viral family including species known to cause widespread arthropod disease. It was detected in samples from Argentina, Australia and New Zealand. Argentine ants in New Zealand were also infected with a strain of Deformed wing virus common to local hymenopteran species, which is a major pathogen widely associated with honeybee mortality. Evidence for active replication of viral RNA was apparent for both viruses. Our results suggest co-introduction and exchange of pathogens within local hymenopteran communities. These viral species may contribute to the collapse of Argentine ant populations and offer new options for the control of a globally widespread invader.     

A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity

Ras family GTPases (RFGs) are known to share many regulatory and effector proteins. How signaling and biological specificity is achieved is poorly understood. Using a proteomics approach, we have identified a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific M-Ras effector. M-Ras targets Shoc2-PP1c to stimulate Raf activity by dephosphorylating the S259 inhibitory site of Raf proteins bound to other molecules of M-Ras or Ras. Therefore, distinct RFGs, through independent effectors, can regulate different steps in the activation of Raf kinases. Shoc2 function is essential for activation of the MAPK pathway by growth factors. Furthermore, in tumor cells with Ras gene mutations, inhibition of Shoc2 expression inhibits MAPK, but not PI3K activity. We propose that the Shoc2-PP1c holoenzyme provides an attractive therapeutic target for inhibition of the MAPK pathway in cancer.     

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane