Activation of oxidative stress response by hydroxyl substituted chalcones and cyclic chalcone analogues in mitochondria.

We investigated the effect of hydroxyl substituted chalcone (1a) and some chalcone analogues (1b-d) on isolated rat liver mitochondria to gain new insights into the cytotoxic mechanism of these compounds. We observed an inhibitory effect on phosphorylation and the partial uncoupling of compounds 1a and 1d. Increased radical generation and possible covalent interaction of the compounds with cellular thiols resulted in glutathione (GSH) depletion and modulation of the investigated mitochondrial activities. Disruption of interconnected mechanisms as electron transport chain and energetic metabolism, ROS production and insufficiency of antioxidant defensive system could lead to induction of cell death.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

Comparison of effects of some cyclic chalcone analogues on selected mitochondrial functions.

In earlier studies, cytotoxity of chalcones (1) and cyclic chalcone analogues E-2-arylidene-tetralones (2) and -benzosuberones (3) towards various murine and human tumour cells has been tested. Preliminary biochemical investigations showed the compounds to inhibit protein and DNA syntheses. It was also found that the compounds affect the cellular thiol status of the treated cells. In order to gain new insights into the cytotoxic mechanism of the compounds effects of some previously investigated 2 and 3 derivatives on isolated rat liver mitochondria was investigated. It was found that the most cytotoxic compounds 2c and 3b significantly decreased the GSH level of the mitochondria. Incubation of the investigated chalcones with reduced GSH under cell-free conditions indicated spontaneous conjugation (non-redox) reaction at pH 7.4 and pH 9.0. Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose showed the compounds to display hydroxyl radical scavenger activity. Investigation of respiratory control ratio of 2c and 3b showed the compounds to display an inhibitory effect on respiration, compound 2b, however, displayed rather an uncoupling effect. The experiments provide further details of cytotoxic effects of the synthetic chalcones displaying dual - cytotoxic and cytoprotective - effects.

A 1-Mb PAC contig spanning the common eliminated region 1 (CER1) in microcell hybrid-derived SCID tumors.

We have developed an elimination test to identify chromosomal regions that contain tumor inhibitory genes. Monochromosomal human/mouse microcell hybrids are generated and passaged through SCID mice. Derived tumors are then analyzed for deletions on the transgenomic chromosome. Using this strategy, we have previously identified a 1.6-cM common eliminated region 1 (CER1) on human 3p21. 3. We now report that CER1 contains 14 markers that are deleted in 19 SCID-derived tumors. A 1-Mb PAC contig that spans CER1 was assembled. Five chemokine receptor genes (CCR1, CCR3, CCR2, CCR5, and CCR6) were localized in CER1 in a 225-kb cluster. The lactotransferrin gene (LTF, or lactoferrin, LF), which reportedly has tumor inhibitory activity, also maps to CER1. Our results create a basis for characterization and further functional testing of genes within CER1.

hCG and the free beta-subunit as screening tests for Down syndrome.

Published studies have reached varying conclusions as to the benefit of replacing human chorionic gonadotropin (hCG) measurements with the free beta-subunit of hCG (the free beta-subunit) for Down syndrome screening. One study reports 14 per cent higher detection for the free beta-subunit, while another finds an actual loss in detection. To explore this issue further, we directly compared the screening performance of hCG and the free beta-subunit, alone and in combination with other serum markers, using banked sera obtained prior to amniocentesis and karyotyping. Altogether, 52 Down syndrome and 5065 unaffected pregnancies were studied. Sera were thawed and assayed for hCG and the free beta-subunit over 1 year. At a 5 per cent false-positive rate, the detection rate for hCG in combination with maternal age and alpha-fetoprotein was higher than when the free beta-subunit was substituted (62 versus 57 per cent). Ultrasound dating and adding unconjugated oestriol both increased detection. The present findings, along with those from six case control studies (our re-analysis), indicate that the screening performances of hCG and the free beta-subunit are similar (median change in detection 0, range -8 to +3 per cent). Under optimal sample collection and transportation conditions, laboratories can expect to achieve similar screening performance using either hCG or the free beta-subunit measurements.