Synthesis of N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides as new selective ligands for sigma receptors

Novel benzofuran-2-carboxamide ligands, which are selective for sigma receptors, have been synthesized via a microwave-assisted Perkin rearrangement reaction and a modified Finkelstein halogen-exchange used to facilitate N-alkylation. The ligands synthesized are the 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides (KSCM-1, KSCM-5 and KSCM-11). The benzofuran-2-carboxamide structure was N-arylated and N-alkylated to include both N-phenyl and N-(3-(piperidin-1-yl)propyl substituents, respectively. These new carboxamides exhibit high affinity at the sigma-1 receptor with K_i values ranging from 7.8 to 34 nM. Ligand KSCM-1 with two methoxy substituents at C-5 and C-6 of the benzofuran ring, and K_i = 27.5 nM at sigma-1 was found to be more selective for sigma-1 over sigma-2.     

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.     

Dream Interpretation with Heterosexual Dating Couples

We examined the effectiveness of the Hill cognitive–experiential model of dream interpretation for working with male and female partners in heterosexual dating couples. Results showed that female partners who received dream interpretation (N = 20) had greater improvements in relationship well-being, insight, and gains from dream interpretation than female partners in the wait-list control group (N = 20). However, male partners who received dream interpretation (N = 20) did not make significant improvements as compared to male partners who did not receive dream interpretation (N = 20). Hence, couples dream interpretation may be more helpful for women than men. Greater effort may be needed to involve men in couples dream sessions in hopes that they will show more gains in relationship well-being and insight.     

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA–protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA–streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilzation of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA ~1000-fold. For example, <0.1 amol/µl (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.     

Ethylene inhibitors promote in vitro regeneration of cowpea (Vigna unguiculata L.)

Summary Ethylene is a plant growth regulator that is known to influence in vitro morphogenesis. This study investigated the effects of three ethylene inhibitors, silver nitrate (AgNO₃), 2,5-norbornadiene, and cobalt chloride (CoCl₂), on the regeneration of cowpea from cotyledon explants. Significant increases in the percentage of regeneration occurred as a result of adding either 50 μM AgNO₃ or 100 μM 2,5-norbornadiene. The number of shoots produced per explant was enhanced by adding 25 μM CoCl₂ or 100 μM norbornadiene. Maximum shoot elongation was obtained with 25 μM of either CoCl₂ or norbornadiene. The effect of the duration of exposure to AgNO₃ was also determined. The greatest percent regeneration was obtained with the addition of 60 μM AgNO₃ either to both the initiation and regeneration stages, or to only the regeneration stage. The promotive effects on organogenesis in response to ethylene inhibitors suggests an important role for ethylene in the process of in vitro morphogenesis of cowpea and may contribute to its normally low regeneration frequency.     

Compositional Discrimination of Decompression and Decomposition Gas Bubbles in Bycaught Seals and Dolphins

Gas bubbles in marine mammals entangled and drowned in gillnets have been previously described by computed tomography, gross examination and histopathology. The absence of bacteria or autolytic changes in the tissues of those animals suggested that the gas was produced peri- or post-mortem by a fast decompression, probably by quickly hauling animals entangled in the net at depth to the surface. Gas composition analysis and gas scoring are two new diagnostic tools available to distinguish gas embolisms from putrefaction gases. With this goal, these methods have been successfully applied to pathological studies of marine mammals. In this study, we characterized the flux and composition of the gas bubbles from bycaught marine mammals in anchored sink gillnets and bottom otter trawls. We compared these data with marine mammals stranded on Cape Cod, MA, USA. Fresh animals or with moderate decomposition (decomposition scores of 2 and 3) were prioritized. Results showed that bycaught animals presented with significantly higher gas scores than stranded animals. Gas composition analyses indicate that gas was formed by decompression, confirming the decompression hypothesis.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

The influence of age and sex on the vocal repertoire of the Antillean manatee (Trichechus manatus manatus) and their responses to call playback

Limited information is available regarding the acoustic communication of Antillean manatees, however, studies have shown that other manatee taxa produce vocalizations as a method of individual recognition and communication. Here, the acoustic signals of 15 Antillean manatees in captivity were recorded, aiming to (1) describe their acoustic repertoire, (2) investigate the influence of sex and age on vocalization, and (3) examine manatee responses to call playback. Six acoustic signals ranging in mean fundamental frequencies from 0.64 kHz to 5.23 kHz were identified: squeaks and screeches were common to adult males, adult females, and juveniles; trills were common to adult males and females; whines were specific to males; creaks were specific to females; and rubbing was specific to juveniles. The structure of squeak vocalizations was significantly different between age and sex classes and screech structure was significantly different between age classes. Squeaks and screeches produced by juveniles had higher frequencies of maximum energy when compared to those produced by adult males and females. A significant increase in the vocalization rate following vocalization playbacks was found for all three age/sex groups. Our results introduce the potential of using acoustic signals in identifying and noninvasively monitoring manatees in the wild in Brazil.