Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX)

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.     

Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P heterozygotes were identified among 9,255 individuals from the general population and had reduced levels of apoB-containing lipoproteins. Most surprisingly, R3480P LDL bound with lower affinity to the LDL receptor than non-carrier LDL in vitro, and these results were confirmed by turnover studies of LDL in vivo. In very low density lipoprotein (VLDL) turnover studies, the amount of VLDL converted to LDL in R3480P heterozygotes was substantially reduced, suggesting that this was the explanation for the hypobetalipoproteinemia observed in these individuals. Our findings emphasized the importance of combining in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context.     

Insect fragments in flour: relationship to lesser grain borer (Coleoptera: Bostrichidae) infestation level in wheat and rapid detection using near-infrared spectroscopy

We determined that the number of insect fragments, quantified using the standard flotation method, in flour milled from wheat infested with larvae, pupae, or preemergent adults of the lesser grain borer, Rhyzopertha dominica (F.), was proportional to infestation level. Wheat infested with a single preemergent adult contributed 28 and 10x as many fragments as wheat infested with a single larva or pupa, respectively. Using regression models that were developed from these data, we predicted that the maximum infestation level that would result in flour with fragment counts below the Food and Drug Administration defect action level (75 fragments/50 g of flour) was 0.95 and 1.5% (380-640 infested kernels/kg of wheat) for pupae and larvae, but it decreased to 0.05% (20 infested kernels/kg) when the grain was infested with preemergent adults. We also reexamined the accuracy and sensitivity of near-infrared spectroscopy (NIRS) for detecting insect fragments in flour by testing three different NIR spectrometers. NIRS-predicted numbers of insect fragments were correlated with the actual number of fragments. NIRS is less precise than the standard flotation method, but it is rapid, nondestructive, does not require extensive sample preparation, and could easily be automated for a more sophisticated sampling protocol for flour based on prescreening samples with NIRS followed up by use of the standard flotation method when necessary.     

GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana

Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP enhancer trap lines were screened and two lines were isolated with GAL4 expression in root xylem-pole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two enhancer trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the enhancer trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong enhancer in the promoter of the LOB-domain protein gene LBD16.