New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.     

Liposomes containing glucosyl ceramide specifically bind T4 bacteriophage: a self-assembling nanocarrier formulation

A unique formulation is described comprising liposomes containing glucosyl ceramide (GluCer) in the lipid bilayer to which bacteriophage T4 was attached. Binding of the phage T4 did not occur to glycolipids, such as galactosyl ceramide, containing an aldose in which the C-2 or C-4 conformations were not identical to glucose. These results strongly support previous proposals that glucose is a major receptor moiety for T4 binding to Escherichia coli. By using the binding of T4 to liposomal GluCer, we further describe a formulation that can be used as a self-assembling combined antigen and adjuvant carrier. A peptide antigen derived from C-trimer (Ct) of HIV-1 gp41 was fused to the highly antigenic outer capsid protein (Hoc), a nonessential protein of T4 that spontaneously binds to the T4 capsid. This resulted in display of the Ct-Hoc construct on the T4 capsid, and specific binding of a human monoclonal antibody that recognizes a peptide sequence of Ct was demonstrated. Liposomes containing monophosphoryl lipid A (MPLA) have been demonstrated to have potent adjuvant activities for experimental vaccines both in humans and animals, and because of this, mice were immunized with the Ct-Hoc-T4 construct that was bound to liposomes containing both GluCer and MPLA, resulting in the induction of high titers of Ct-specific antibodies. We conclude that liposomes containing both GluCer and MPLA can spontaneously bind to a construct of T4 that displays antigens that spontaneously binds to the capsid of T4 bacteriophage. This formulation could be utilized as an easily manufactured self-assembling antigen and adjuvant carrier.     

SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus

EDS1, PAD4, and SAG101 are common regulators of plant immunity against many pathogens. EDS1 interacts with both PAD4 and SAG101 but direct interaction between PAD4 and SAG101 has not been detected, leading to the suggestion that the EDS1-PAD4 and EDS1-SAG101 complexes are distinct. We show that EDS1, PAD4, and SAG101 are present in a single complex in planta. While this complex is preferentially nuclear localized, it can be redirected to the cytoplasm in the presence of an extranuclear form of EDS1. PAD4 and SAG101 can in turn, regulate the subcellular localization of EDS1. We also show that the Arabidopsis genome encodes two functionally redundant isoforms of EDS1, either of which can form ternary complexes with PAD4 and SAG101. Simultaneous mutations in both EDS1 isoforms are essential to abrogate resistance (R) protein-mediated defense against turnip crinkle virus (TCV) as well as avrRps4 expressing Pseudomonas syringae. Interestingly, unlike its function as a PAD4 substitute in bacterial resistance, SAG101 is required for R-mediated resistance to TCV, thus implicating a role for the ternary complex in this defense response. However, only EDS1 is required for HRT-mediated HR to TCV, while only PAD4 is required for SA-dependent induction of HRT. Together, these results suggest that EDS1, PAD4 and SAG101 also perform independent functions in HRT-mediated resistance.     

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; P_value = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; P_value = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; P_value = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.