Voltage sensitivities and deactivation kinetics of histamine H3 and H4 receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms of the human histamine H₃ receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H₄ receptor, which is expressed mainly on hematopoietic cells. We found that the hH₃⁴⁴⁵ receptor is the most sensitive to voltage, whereas the hH₃³⁶⁵ and H₄ receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms, with the hH₃³⁶⁵ isoform being five to six-fold slower than the hH₃⁴⁴⁵ receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH₄ receptor. The differences in voltage sensitivities and deactivation kinetics between the hH₃⁴⁴⁵, hH₃³⁶⁵, and H₄ receptors might be relevant to their respective physiological roles.     

Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

Drug–membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution ¹H and ¹³C nuclear magnetic resonance (NMR) and solid state ¹³C and ³¹P (NMR) spectroscopies. In addition, ³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT₁ receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.     

Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α₂β₂ tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.     

LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.     

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that?the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked?by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in?vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.     

Innate immune signalling at the intestinal epithelium in homeostasis and disease

The intestinal epithelium-which constitutes the interface between the enteric microbiota and host tissues-actively contributes to the maintenance of mucosal homeostasis and defends against pathogenic microbes. The recognition of conserved microbial products by cytosolic or transmembrane pattern recognition receptors in epithelial cells initiates signal transduction and influences effector cell function. However, the signalling pathways, effector molecules and regulatory mechanisms involved are not yet fully understood, and the functional outcome is poorly defined. This review analyses the complex and dynamic role of intestinal epithelial innate immune recognition and signalling, on the basis of results in intestinal epithelial cell-specific transgene or gene-deficient animals. This approach identifies specific epithelial cell functions within the diverse cellular composition of the mucosal tissue, in the presence of the complex and dynamic gut microbiota. These insights have thus provided a more comprehensive understanding of the role of the intestinal epithelium in innate immunity during homeostasis and disease.     

Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond

Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.     

Modelling chlorophyll fluorescence of kiwi fruit (Actinidia deliciosa)

Kiwi fruit displays chlorophyll fluorescence. A physical model was developed to reproduce the observed original fluorescence for the whole fruit, from the emission of the different parts of the kiwi fruit. The spectral distribution of fluorescence in each part of the fruit, was corrected to eliminate distortions due to light re-absorption and it was analyzed in relation to photosystem II-photosystem I ratio. Kiwi fruit also displays variable chlorophyll-fluorescence, similar to that observed from leaves. The maximum quantum efficiency of photosystem II photochemistry (F(v)/F(m)), the quantum efficiency of photosystem II (Φ(PSII)), and the photochemical and non-photochemical quenching coefficients (q(P) and q(NP) respectively) were determined and discussed in terms of the model developed. The study was extended by determining the photosynthetic parameters as a function of the storage time, at both 4 °C and room temperature for 25 days.