A novel substance P binding site in rat brain regions modulates TRH receptor binding

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [³H](2-Me-His³)TRH ([³H]MeTRH) on membranes from rat brain regions at 0°C for 5 h. Amygdaloid membranes bound [³H]MeTRH with high-affinity (K _d=3.1±0.5 nM (n=4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC₅₀ for SP=65 M). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP>nonapeptide (3–11)>hexapeptide (6–11)>heptapeptide (5–11)>pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [³H]MeTRH binding in hippocampus> spinal cord>retina>n. accumbens>hypothalamus>amygdaloid>olfactory bulb pituitary>pons/medulla in parallel assays. In amygdaloid membranes SP (50 M) reduced the apparent maximum receptor density by 39% (p<0.01) without altering the binding affinity, and 100 M SP induced a biphasic dissociation of [³H]MeTRH with kinetics faster than those induced by both TRH (10 M) and serotonin (100 M). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [³H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.A preliminary account of these studies has been presented to the British Pharmacological Society (9).     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker

Biochemical Genetics - Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla...     

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.     

RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genome-wide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)- and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.     

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [125I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores, as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.     

Identification of Small Molecule and Genetic Modulators of AON-Induced Dystrophin Exon Skipping by High-Throughput Screening

One therapeutic approach to Duchenne Muscular Dystrophy (DMD) recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD), by employing antisense oligonucleotides (AONs) targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened ∼10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2) were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.     

Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl₂ and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.     

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

Background

For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.

Results

Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.

Conclusions

In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.