Deleterious effects of endogenous and exogenous testosterone on mesenchymal stem cell VEGF production

Modulating the paracrine effects of bone marrow mesenchymal stem cells (BMSCs) may be important for the treatment of ischemic myocardial tissue. In this regard, endogenous estrogen may enhance BMSC vascular endothelial growth factor (VEGF) production. However, little information exists regarding the effect of testosterone on stem cell function. We hypothesized that 1) endogenous or exogenous estrogen will enhance stem cell production of VEGF and 2) endogenous or exogenous testosterone will inhibit BMSC VEGF production. BMSCs were collected from adult male, female, castrated male, and ovariectomized female rats. One hundred thousand cells were incubated with testosterone (1, 10, or 100 nM) or estrogen (0.15, 1.5, or 15 nM) for 48 h. Cell supernatants were collected, and VEGF was measured by ELISA. BMSCs harvested from castrated males, normal females, and ovariectomized females produced more VEGF compared with normal males. Castration was associated with the highest level (1,018 +/- 98.26 pg/ml) of VEGF production by BMSCs, which was significantly more than that produced by BMSCs harvested from normal male and normal female animals. Exogenous testosterone significantly reduced VEGF production in BMSCs harvested from ovariectomized females in a dose-dependent manner. Exogenous estrogen did not alter BMSC VEGF production. These findings suggest that testosterone may work on BMSCs to decrease protective growth factor production and that effective removal of testosterone's deleterious effects via castration may prove to be beneficial in terms of protective factor production. By manipulating the mechanisms that BMSCs use to produce growth factors, we may be able to engineer stem cells to produce maximum growth factors during therapeutic use.     

Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts.

BACKGROUND/AIMS: Endothelin-1 (ET-1) has been implicated in pathologic remodelling and tissue repair processes in the heart. We investigated the effects of ET-1 on growth and collagen synthesis responses in cardiac fibroblasts isolated from human hearts. We also studied the receptor subtype(s) mediating such responses and the factors regulating their expression. METHODS: Fibroblasts were isolated from cardiac transplant recipient hearts and characterised by immunocytochemistry. Serum-starved cells were exposed to ET-1 and incorporation of [3H]proline and thymidine were measured as indexes of collagen and DNA synthesis respectively. Blocking experiments utilised the selective ETA receptor antagonist BQ123 and the ETB antagonist BQ788. RESULTS: ET-1 elicited a potent collagen synthesis response in cardiac fibroblasts, with a maximum 29+/-5% increase that was abolished by BQ123. Cardiac fibroblasts responded to ET-1 with a concentration-dependent decrease in DNA synthesis rate. The effects of ET-1 were similar to those of TGF-beta. Radioligand binding studies revealed the presence of high-affinity ET-1 binding sites on these cells, which were upregulated by treatment with the growth factors PDGF and EGF but downregulated by TGF-beta. CONCLUSIONS: These results therefore implicate ET-1 as a trophic agent in the human heart with the ability to influence the development of cardiac fibrosis.     

DNA barcoding and the documentation of alien species establishment on sub-Antarctic Marion Island

Invasive alien species constitute a substantial conservation challenge in the terrestrial sub-Antarctic. Management plans, for many of the islands in the region, call for the prevention, early detection, and management of such alien species. However, such management may be confounded by difficulties of identification of immatures, especially of holometabolous insects. Here we show how a DNA barcoding approach has helped to overcome such a problem associated with the likely establishment of an alien moth species on Marion Island. The discovery of unidentifiable immatures of a noctuid moth species, 5 km from the research station, suggested that a new moth species had colonized the island. Efforts to identify the larvae by conventional means or by rearing to the adult stage failed. However, sequencing of 617 bp of the mitochondrial cytochrome oxidase subunit I gene, and comparison of the sequence data with sequences on GENBANK and the barcoding of life database enabled us to identify the species as Agrotis ipsilon (Hufnagel), a species of which adults had previously been found regularly at the research station. Discovery of immatures of this species, some distance from the research station, suggests that a population may have established. It is recommended that steps to be taken to eradicate the species from Marion Island.     

Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 μl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their ‘on–off’ signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1 × 10⁴ improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.     

Regulation of somatic firing dynamics by backpropagating dendritic spikes

Pyramidal cells of the apteronotid ELL have been shown to display a characteristic mechanism of burst discharge, which has been shown to play an important role in sensory coding. This form of bursting depends on a reciprocal dendro-somatic interaction, in which discharge of a somatic spike causes a dendritic spike, which in turn contributes a dendro-somatic current flow to create a depolarizing afterpotential (DAP) in the soma. We review here our recent work showing how the timing of this DAP influences the somatic firing dynamics, and how the degree of inactivation of dendritic Na⁺ currents can cause an increased delay between somatic and dendritic spikes. This ultimately allows the DAP to become more effective at increasing the excitability of the somatic spike generating mechanism. Further, this delay between dendritic and somatic spiking can be regulated by strongly hyperpolarizing GABA_B mediated dendritic inhibition, allowing the burst dynamics to fall under synaptic regulation. In contrast, a weaker, shunting inhibition due to GABA_A mediated dendritic inhibition can regulate the dendritic spike waveform to decrease the dendro-somatic current flow and the resulting DAP. We therefore show that the qualitative behaviour of an individual cell can depend on the degree of synaptic input, and the exact timing of events across the spatial extent of the neuron. Thus, our results serve to illustrate the complex dynamics that can be observed in cells with significant dendritic arborisation, a nearly ubiquitous adaptation amongst principal neurons.     

Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning

Sonic hedgehog (Shh) ligand secreted by the notochord induces distinct ventral cell identities in the adjacent neural tube by a concentration-dependent mechanism. To study this process, we genetically engineered mice that produce bioactive, fluorescently labeled Shh from the endogenous locus. We show that Shh ligand concentrates in close association with the apically positioned basal body of neural target cells, forming a dynamic, punctate gradient in the ventral neural tube. Both ligand lipidation and target field response influence the gradient profile, but not the ability of Shh to concentrate around the basal body. Further, subcellular analysis suggests that Shh from the notochord might traffic into the neural target field by means of an apical-to-basal-oriented microtubule scaffold. This study, in which we directly observe, measure, localize and modify notochord-derived Shh ligand in the context of neural patterning, provides several new insights into mechanisms of Shh morphogen action.