Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.     

The static magnetic field effects on ouabain H3 binding by cancer tissue

Radioactive labeled ouabain was used for estimating the static magnetic field (SMF) induced cell volume changes. Ouabain is a specific inhibitor of Na+/K+ ATPase, and can be used for estimating its quantity--thus giving information about the cell volume changes. Ouabain binding by cancer and normal glandular tissues of breast cancer patients and normal glandular tissues of healthy women was measured after exposure of tissues to SMF 0.2T. SMF exposure led to a decrease of ouabain binding in both normal and cancer tissues when ouabain concentration in the external medium was 10(-9) M, while in the case of higher concentrations of ouabain (10(-7) M, 10(-6) M) an increase of ouabain binding was seen. The normal glandular tissues of healthy women were sensitive to SMF only at the highest concentrations of ouabain used in our experiments. The SMF-induced decrease of binding at low ouabain concentrations was considered as an evidence for the dehydration effect of SMF. It is suggested that the SMF could influence the cancer cell metabolism through cell hydration changes.     

EEG reactions during creative activity

Evoked activity in response to light was recorded in students performing a verbal creative task. The changes in the amplitude of the N200 negative component of evoked potentials were analyzed. The amplitude of the N200 component was significantly increased in the frontal and anterior frontal areas of the left hemisphere, which indicated increased activity in the cortical structures involved in divergent thinking. The amplitude of the N200 component was increased in the temporo-parieto-occipital area of the right hemisphere, which indicated that the posterior associative region of the right hemisphere was also involved in the creative activity. The data obtained suggest that the frontal and temporo-parieto-occipital areas of the cerebral cortex actively participate in the performance of a creative test with distinct elements of complexity.     

Hypericum alpestre extract exhibits in vitro and in vivo anticancer properties by regulating the cellular antioxidant system and metabolic pathway of L-arginine

Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the  l -arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG-hydroxy-nor- l -arginine and NO inhibitor NG-nitro-Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12-dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro-oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF-7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC-Q-Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer-like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with N^ω-OH-nor- l -arginine and N^ω-nitro- l -arginine methyl ester, H. alpestre extract exhibits pro- and antioxidant, antiangiogenic, and cytotoxic effects.     

The location of the maximum amplitude focus of cortical evoked potential component N200 in subjects solving a maze problem

The dependence of the efficiency of maze problem solution on the “commitment” of the frontal, parietal, temporal, and occipital cortices to the activity was studied. For this purpose, topographic neurophysiological characteristics of component N200 of cortical evoked potentials were determined and used to locate the maximum amplitude focus (MAF). Right-and left-handed subjects of both sexes (50 persons aged 18–23 years) were divided into five groups: (I) women with a low nonverbal IQ, (II) women with a high nonverbal IQ, (III) men with a low nonverbal IQ, (IV) men with a high nonverbal IQ, and (V) left-handed men with a high nonverbal IQ. In subjects from group III, a successful solution of a maze problem was associated with a high activity only in the occipital cortex. In groups II and V, in addition to the strong commitment of the occipital cortex, about the same activity was observed in the frontal cortex. In groups I and IV, and MAF appeared irregularly if at all, which may have accounted for the low efficiency of maze problem solution in these subjects.     

Spatial organization of receptive fields and dynamic peculiarities of neurons of the extrastriate area 21a of the cat cerebral cortex

In neurons of the extrastriate area 21a of the cat cortex, we examined the mode of initiation and peculiarities of inhibitory components in responses of these units to visual stimulation. About 31% of the studied neurons generated complex responses to mobile visual stimuli; the parameters of inhibitory components in these responses (location and duration) were different and depended on the contrast, dimension, and shape of the visual stimuli presented. We compared in detail the stationary spatial organization of receptive fields (RFs) and parameters of neuronal responses to presentation of moving stimuli in order to estimate the correlation between static and dynamic characteristics of the activity generated by the studied neurons. Our experiments showed that in most cases the neurons possessing identical homogeneous static characteristics of the RFs with off, on-off, and on responses could demonstrate quite different patterns of responses to moving stimuli, which differed from each other both in localization of inhibitory zones and discharge centers within the RFs and in time parameters of the components of these responses. The obtained data allow us to hypothesize that the dynamic characteristics of visually sensitive neurons in the extrastriate associative cortical regions are formed due to complex processes of spatial interaction between their “classic” RFs and the surrounding visual space. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 119–129, March–April, 2008.