Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.     

Habitat preferences of lesser rheas in Argentine Patagonia

This work reports the first results of a 3-year study (1998–2001) on habitat use and preferences by wild lesser rheas (Rhea pennata pennata) in the ecotone Monte–Patagonian steppe of Argentina. Ponds and four different habitat alternatives for lesser rheas were determined using satellite images and different structural vegetation characteristics: steppe, shrubland, shrub steppe, and mallines. Lesser rheas (adults, juveniles and chicks) used all habitats available to feed, although they showed preference for mallines, open areas that offer good visibility and abundant food resource. Rheas used shrub steppe and steppe for nesting, but they did not show preference for either habitat type. Nest site had a higher percentage of vegetation cover than control sites. Lesser rheas apparently preferred concealed sites for nesting since they offer protection from severe climate conditions and from predators. Our results suggest that habitat preference by lesser rheas counterbalances profitability of feeding to the corresponding cost of predation.     

Synthesis and in vitro sodium channel blocking activity evaluation of novel homochiral mexiletine analogs

New chiral mexiletine analogs were synthesized in their optically active forms and evaluated in vitro as use‐dependent blockers of skeletal muscle sodium channels. Tests carried out on sodium currents of single muscle fibers of Rana esculenta demonstrated that all of them exerted a higher use‐dependent block than mexiletine. The most potent analog, (S)‐3‐(2,6‐dimethylphenoxy)‐1‐phenylpropan‐1‐amine (S)‐( 5 ), was six‐fold more potent than (R)‐Mex in producing a tonic block. As observed with mexiletine, the newly synthesized compounds exhibit modest enantioselective behavior, that is more evident in 3‐(2,6‐dimethylphenoxy)butan‐1‐amine ( 3 ). Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Light trapping of biting midges Culicoides spp. (Diptera: Ceratopogonidae) with green light-emitting diodes

Abstract  A study of responses by Culicoides spp. to light traps with light-emitting diodes (LEDs) confirmed that the livestock virus vector, Culicoides brevitarsis Kieffer, was preferentially attracted to green light. Four species shown previously to respond to blue light exhibited significantly higher responses to ultraviolet (UV) light. Field trials comparing green LEDs with incandescent lights in New South Wales, Northern Territory and East Timor confirmed the superiority of green LEDs for catching C. brevitarsis . The green LED traps also had a significant advantage over incandescent traps for a wide range of Culicoides species. These included: species whose ultimate preference was for UV, most species that are proven or potential vectors of viruses affecting Australian livestock and native animals and the main species affecting humans in northern Australia. Use of green LEDs has been adopted for trapping C. brevitarsis , especially in its marginal and low-density areas, and for detecting incursions of new Culicoides spp. into Australia. Use could be possible for species in other insect Orders.     

Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.     

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer

Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2′-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.     

Collation and data-mining of literature bioactivity data for drug discovery

The challenge of translating the huge amount of genomic and biochemical data into new drugs is a costly and challenging task. Historically, there has been comparatively little focus on linking the biochemical and chemical worlds. To address this need, we have developed ChEMBL, an online resource of small-molecule SAR (structure-activity relationship) data, which can be used to support chemical biology, lead discovery and target selection in drug discovery. The database contains the abstracted structures, properties and biological activities for over 700000 distinct compounds and in excess of more than 3 million bioactivity records abstracted from over 40000 publications. Additional public domain resources can be readily integrated into the same data model (e.g. PubChem BioAssay data). The compounds in ChEMBL are largely extracted from the primary medicinal chemistry literature, and are therefore usually 'drug-like' or 'lead-like' small molecules with full experimental context. The data cover a significant fraction of the discovery of modern drugs, and are useful in a wide range of drug design and discovery tasks. In addition to the compound data, ChEMBL also contains information for over 8000 protein, cell line and whole-organism 'targets', with over 4000 of those being proteins linked to their underlying genes. The database is searchable both chemically, using an interactive compound sketch tool, protein sequences, family hierarchies, SMILES strings, compound research codes and key words, and biologically, using a variety of gene identifiers, protein sequence similarity and protein families. The information retrieved can then be readily filtered and downloaded into various formats. ChEMBL can be accessed online at https://www.ebi.ac.uk/chembldb.     

Distinct immunomodulatory properties of Lactobacillus paracasei strains

Aims: This study was performed to ascertain the immunomodulatory effect of Lactobacillus paracasei strains. These strains were also genetically characterized. Methods and Results: The strains were genetically differentiated by using the fluorescent‐amplified fragment length polymorphism technique, which led to the identification of several molecular markers unique to each strain. To determine the immunomodulatory properties, we evaluated the effect of strains on dendritic cell maturation, dextran uptake, ability to induce proliferation of allogenic T cells and cytokine secretion. The results indicated that all the strains stimulated phenotypic maturation of dendritic cells (DCs), but they acted differently on DCs in relation to the other tested properties; notably, a different effect on cytokine secretion was detected. Conclusions: The results of this study revealed different immunomodulatory properties of strains of the species Lact. paracasei. Strain IMPC 4.1 showed an interesting anti‐inflammatory ability. Probiotic strains IMPC 2.1 and LMG P‐17806 were characterized by a similar and intermediate ability to induce cytokine secretion in contrast to the very low ability of strain LMG 23554. Significance and Impact of Study: Our results confirm that each single strain of a bacterial species appears to influence the immune system in a peculiar manner. The evaluation of the different types and/or levels of cytokines whose secretion is induced by each strain could be relevant to define its pro‐ or anti‐inflammatory properties and its more appropriate clinical use.