Effects of 9-ene-tetrahydrocannabinol on expression of β-type transforming growth factors, insulin-like growth factor-I and c-myc genes in the mouse uterus

Effects of cannabinoid on expression of β-type transforming growth factors (TGF-β1, -β2 and -β3), insulin-like growth factor-I (IGF-I) and c-myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17β (E₂) and/or progesterone (P₄), and uteri were analyzed at various times thereafter. TGF-β isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E₂ caused a modest increase in TGF-β1 and -β3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E₂ by changing the timing for the peak of TGF-β3 mRNA levels to 12 h. In comparison, E₂ treatment substantially elevated the levels of TGF-β2 mRNA at 6 h, and THC potentiated this E₂ response without affecting the timing for the response. Imposition of P₄ treatment did not antagonize any of these responses. P₄ treatment alone or with THC had insignificant effects on mRNA levels for these TGF-β isoforms. Uterine levels of IGF-I and c-myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E₂ treatment induced a rapid, but transient, increase in IGF-I and c-myc mRNAs, and THC antagonized the rapid c-myc mRNA response and altered the timing of the IGF-I mRNA response. P₄ treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P₄ plus E₂ resulted in further modest increases in IGF-I and c-myc mRNA levels as compared to E₂ or P₄ treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression.     

Effect of grazing removal on aboveground vegetation and soil seed bank composition in sub-alpine grasslands of northern Iran

Background: Knowledge about vegetation and soil seed bank composition and the processes that contribute to vegetation recovery after the removal of heavy grazing is lacking in sub-alpine ecosystems.

Aims: In order to assess the effects of large herbivores on above-ground vegetation (AGV) and soil seed bank (SSB) characteristics, intensively sheep-grazed areas were compared to adjacent areas where grazing had been removed 10 years previously in a sub-alpine grassland of northern Iran.

Methods: A total of 40 4-m² (2 m × 2 m) plots were established in each treatment, and soil samples were collected from each plot within a depth of 10 cm. Plant species composition was determined for each plot during the flowering stage of herbaceous species in June 2011. The seedling emergence method was used to estimate SSB composition.

Results: A total of 45 species (23 annuals and 22 perennials) emerged from the soil samples of the grazed area, while the number of species emerged from the soil samples of the previously grazing area was 76 (37 annuals and 39 perennials). The removal of grazing led to a significant increase in species richness and seed density in the SSB. Species turnover of AGV was higher, and that of the SSB was similar for grazed areas compared with areas that were formerly grazed. Detrended correspondence analysis ordination of AGV composition showed a clearly separate structure between grazed plots and plots where grazing has been removed, while the segregation was less clear for SSB composition.

Conclusions: We concluded that restoration of locally degraded sites cannot rely on the SSB when grazing is stopped simultaneously and unvegetated gaps are colonised by vegetative growth rather than by seed.     

An Approach to Design Highly Durable Piezoelectric Nanogenerator Based on Self‐Poled PVDF/AlO‐rGO Flexible Nanocomposite with High Power Density and Energy Conversion Efficiency

Till date, fabrication of piezoelectric nanogenerator (PNG) with highly durable, high power density, and high energy conversion efficiency is of great concern. Here a flexible, sensitive, cost effective hybrid piezoelectric nanogenerator (HPNG) developed by integrating flexible steel woven fabric electrodes into poly(vinylidene fluoride) (PVDF)/aluminum oxides decorated reduced graphene oxide (AlO‐rGO) nanocomposite film is reported where AlO‐rGO acts as nucleating agent for electroactive β‐phase formation. The HPNG exhibits reliable energy harvesting performance with high output, fast charging capability, and high durability compared with previously reported PVDF based PNGs. This HPNG is capable for harvesting energy from a variety and easy accessible biomechanical and mechanical energy sources such as, body movements (e.g., hand folding, jogging, heel pressing, and foot striking, etc.) and machine vibration. The HPNG exhibits high output power density and energy conversion efficiency, facilitating direct light on different color of several commercial light‐emitting diodes instantly and powers up many portable electronic devices like wrist watch, calculator, speaker, and mobile liquid crystal display (LCD) screen through capacitor charging. More importantly, HPNG retains its performance after long compression cycles (≈158 400), demonstrating great promise as a piezoelectric energy harvester toward practical applications in harvesting biomechanical and mechanical energy for self‐powered systems.     

Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state

Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca²⁺ through the interaction of its self-contained Ca²⁺ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca²⁺ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 10⁶ M^-1 and a binding enthalpy change of ?3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity.     

Significant differences between mouse and human trophinins are revealed by their expression patterns and targeted disruption of mouse trophinin gene.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.     

Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-kappaB activation in endothelial cells

Thrombin activation of protease-activated receptor-1 induces Ca(2+) influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca(2+) influx induced by the depletion of Ca(2+) stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca(2+) influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappaB (NF-kappaB) binding sites. Co-expression of dominant negative IkappaBalpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappaB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappaB binding sites located between -1623 and -871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappaB to NF-kappaB consensus sites were located in this domain. Supershift assays using NF-kappaB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca(2+) depletion, buffering of intracellular Ca(2+), or down-regulation of protein kinase Calpha downstream of Ca(2+) influx all blocked thrombin-induced NF-kappaB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca(2+) influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappaB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.     

The Bysl gene product, bystin, is essential for survival of mouse embryos

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin apparently disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.