Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.     

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg? > Arg? ? Arg?. The conjugate with Arg? exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg?-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg?-1 and 17-desacetylvinblastineTrp-Arg?-2 are epimers at the tryptophan α carbon atom.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.     

Hit-to-lead optimization of disubstituted oxadiazoles and tetrazoles as mGluR5 NAMs

Here we report the discovery and early SAR of a series of mGluR5 negative allosteric modulators (NAMs). Starting from a moderately active HTS hit we synthesized 3,5-disubstituted-oxadiazoles and tetrazoles as mGluR5 NAMs. Based on the analysis of ligand efficiency and lipophilic efficiency metrics we identified a promising lead candidate as a starting point for further optimization.     

Development and characterization of a FIA system for selective assay of l-ascorbic acid in food samples

An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration curve in 25–400 μmol/L range was R ² = 0.99.     

The role of high-dose-rate brachytherapy boost in breast-conserving therapy: Long-term results of the Hungarian National Institute of Oncology

AimTo report the long-term results of high-dose-rate (HDR) brachytherapy (BT) boost for breast cancer patients treated with conservative surgery and radiotherapy.Materials and methodsBetween 1995 and 2007, 100 early-stage breast cancer patients received an HDR BT boost after conservative surgery and whole breast irradiation. Ten patients (10%) received a single-fraction HDR boost of 8–10.35 Gy using rigid needles, while 90 (90%) were treated with a fractionated multi-catheter HDR BT boost. The latter consisted of 3 × 4 Gy (n = 19), 3 × 4.75 Gy (n = 70), and 2 × 6.4 Gy (n = 1). Breast cancer related events, cosmetic results and side effects were assessed.ResultsAt a median follow-up time of 94 months (range: 8–152) only 7 (7%) ipsilateral breast failures were observed for a 5- and 8-year actuarial rate of 4.5 and 7.0%, respectively. The 8-year disease-free, cancer-specific, and overall survival was 76.1, 82.8, and 80.4%, respectively. Cosmetic outcome was rated excellent in 17%, good in 39%, fair in 33%, and poor in 11%. Data on late radiation side effects were available for 91 patients (91%). Grade 3 fibrosis and grade 3 telangiectasia occurred in 6 (6.6%) and 2 (2.2%) patients, respectively. In univariate analysis only positive margin status had a significant negative effect on local control.ConclusionsHDR BT boost using multi-catheter implants produce excellent long-term local tumour control with acceptable cosmetic outcome and low rate of grade 3 late radiation side effects.     

Influence of neonatal insulin imprinting on the insulin binding capacity of rat erythrocytes in adulthood

A single neonatal insulin treatment decreased considerably the insulin binding capacity of erythrocytes in adult rats, by analogy of the behaviour of the hepatic insulin receptors in response to insulin exposure during the perinatal period, or during liver regeneration in adulthood. These observations substantitate earlier conclusions on the mechanism of imprinting and strongly suggest the universality of perinatal imprinting in living organisms. In vitro insulin exposure of the erythrocytes of adult rats depressed 48 h later the erythrocytic insulin binding capacity to a similar degree in individuals treated and not treated with insulin when newborn, from which it follows that neonatal exposure had no influence on erythrocytic response to later in vitro treatment. In the light of the present study the use of erythrocytes as model cells for imprinting studies deserves consideration.     

Comparison of micrometer‐ and scanning electron microscope‐based measurements of avian eggshell thickness

ABSTRACT The study of avian eggshell structure, including composition, pigmentation, thickness, and strength, has important ecological and economic implications. Previous investigators have used a variety of techniques to derive either direct measures or indirect estimates of eggshell thickness. Assessing the repeatability and method agreement of different techniques is necessary to permit comparison of eggshell thickness values from different studies on various genetic stocks, populations, and species. We recorded and analyzed measurements of eggshell thickness using two methods, micrometers and scanning electron microscopy (SEM), for several Palaeognathae and Neognathae taxa, including nonpasserines and passerines. Applying a tolerance‐interval approach, we found that repeatability of measurements for eggs with thinner shells (<300 μm, all Neognathae taxa) was worse than for eggs with thicker shells (Palaeognathae taxa), but was still statistically and biologically reasonable given that the relative magnitude of intramethod agreements was <11%. Our results support previous predictions that measurements made using a micrometer are comparable to those made using SEM. This finding is particularly important given the relative ease and cost efficiency of the micrometer method. Importantly, these new analyses can be used to validate the use of published data from previous studies of micrometer‐based eggshell thickness for both intra‐ and interspecific comparisons.     

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.