EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand their role in TKI-resistant NSCLCs, we examined changes in miRNA that are mediated by tyrosine kinase receptors. Here we report that miR-30b, miR-30c, miR-221 and miR-222 are modulated by both epidermal growth factor (EGF) and MET receptors, whereas miR-103 and miR-203 are controlled only by MET. We showed that these miRNAs have important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ? (PKC-?) and sarcoma viral oncogene homolog (SRC). These findings suggest that modulation of specific miRNAs may provide a therapeutic approach for the treatment of NSCLCs.     

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB(2)) receptor. The results were consistent with changes in cAMP content of hCB(2)-transfected human embryonic kidney (HEK) cells challenged with the same CB(2)-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors. ()     

Relaxin-3 and RXFP3 expression, and steroidogenic actions in the ovary of teleost fish

Relaxins are peptides similar in secondary structure to insulins. In teleost genomes, five or six relaxin genes have been identified. Two relaxins group closely with mammalian relaxin-3 on phylogenetic analysis and are named relaxin-3a and b. We refer to the remainder as relaxins c to f. Ovarian expression of relaxin-3a, d and f genes, and the relaxin-3 receptor gene Rxfp3, was studied in Danio rerio using RT-PCR. Immunohistochemistry was used to determine the distribution of relaxin-3 peptides and RXFP3 in the ovary of Fundulus heteroclitus (killifish). Thirdly, enzyme immunoassays and ovarian follicular culture were used to determine the effect of treatment with human recombinant relaxin-3 on the production of 17beta-estradiol and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in killifish ovarian follicles. Relaxin-3a, d, f, and Rxfp3 genes were expressed regardless of sex or reproductive condition. Relaxin-3 immunostaining was present in mid to late follicular stages within cortical alveoli of the oocyte cytopasm, whereas receptor staining was localized to follicular cells. Treatment with relaxin-3 enhanced 17beta-estradiol production in early and late maturing follicles, but did not have an effect in vitellogenic follicles. Relaxin-3 appeared to suppress the release of MIS production. This suggests that relaxin peptides may be involved with estradiol-dependant events in follicular development.     

Interocular yoking in human saccades examined by mutual information analysis

Background

Saccadic eye movements align the two eyes precisely to foveate a target. Trial-by-trial variance of eye movement is always observed within an identical experimental condition. This has often been treated as experimental error without addressing its significance. The present study examined statistical linkages between the two eyes’ movements, namely interocular yoking, for the variance of eye position and velocity.

Methods

Horizontal saccadic movements were recorded from twelve right-eye-dominant subjects while they decided on saccade direction in Go-Only sessions and on both saccade execution and direction in Go/NoGo sessions. We used infrared corneal reflection to record simultaneously and independently the movement of each eye. Quantitative measures of yoking were provided by mutual information analysis of eye position or velocity, which is sensitive to both linear and non-linear relationships between the eyes’ movements. Our mutual information analysis relied on the variance of the eyes movements in each experimental condition. The range of movements for each eye varies for different conditions so yoking was further studied by comparing GO-Only vs. Go/NoGo sessions, leftward vs. rightward saccades.

Results

Mutual information analysis showed that velocity yoking preceded positional yoking. Cognitive load increased trial variances of velocity with no increase in velocity yoking, suggesting that cognitive load may alter neural processes in areas to which oculomotor control is not tightly linked. The comparison between experimental conditions showed that interocular linkage in velocity variance of the right eye lagged that of the left eye during saccades.

Conclusions

We conclude quantitative measure of interocular yoking based on trial-to-trial variance within a condition, as well as variance between conditions, provides a powerful tool for studying the binocular movement mechanism.

     

A step towards non-invasive characterization of the human frontal eye fields of individual subjects

Background

Identifying eye movement related areas in the frontal lobe has a long history, with microstimulation in monkeys producing the most clear-cut results. For humans, however, there is still no consensus about the location and the extent of the frontal eye field (FEF). There is also no simple non-invasive method for unambiguously defining the FEF in individual subjects, a prerequisite for clinical applications. Here we explore the use of magnetoencephalography (MEG) for the non-invasive identification and characterization of FEF activity in an individual subject.

Methods

We mapped human brain activity before, during and after saccades by applying tomographic analysis to MEG data. Statistical parametric maps and circular statistics produced plausible FEF loci, but no unambiguous definition for individual subjects. Here we first computed the spectral decomposition and correlation with electrooculogram (EOG) of the tomographic brain activations. For each of these two measures statistical comparisons were made between different saccades.

Results

In this paper, we first review the frontal cortex activations identified in earlier animal and human studies and place the putative human FEFs in a well-defined anatomical framework. This framework is then used as reference for describing the results of new Fourier analysis of the tomographic solutions comparing active saccade tasks and their controls. The most consistent change in the dorsal frontal cortex was at the putative left FEF, for both saccades to the left and right. The asymmetric result is consistent with the 1-way callosal traffic theory. We also showed that the new correlation analysis had its most consistent change in the contralateral putative FEF. This result was obtained for EOG latencies before saccade onset with delays of a few hundreds of milliseconds (FEF activity leading the EOG) and only for visual cues signaling the execution of a saccade in a previously defined saccade direction.

Conclusions

The FEF definition derived from microstimulation describes only one of the areas in the dorsal lateral frontal lobe that act together to plan, prepare and execute a saccade. The definition and characterization of these areas in an individual subject can be obtained from non-invasive MEG measurements.