Localization of cannabinoid receptors CB1, CB2, GPR55, and PPARα in the canine gastrointestinal tract

The endocannabinoid system (ECS) is composed of cannabinoid receptors, their endogenous ligands, and the enzymes involved in endocannabinoid turnover. Modulating the activity of the ECS may influence a variety of physiological and pathophysiological processes. A growing body of evidence indicates that activation of cannabinoid receptors by endogenous, plant-derived, or synthetic cannabinoids may exert beneficial effects on gastrointestinal inflammation and visceral pain. The present ex vivo study aimed to investigate immunohistochemically the distribution of cannabinoid receptors CB1, CB2, G protein-coupled receptor 55 (GPR55), and peroxisome proliferation activation receptor alpha (PPARα) in the canine gastrointestinal tract. CB1 receptor immunoreactivity was observed in the lamina propria and epithelial cells. CB2 receptor immunoreactivity was expressed by lamina propria mast cells and immunocytes, blood vessels, and smooth muscle cells. Faint CB2 receptor immunoreactivity was also observed in neurons and glial cells of the submucosal plexus. GPR55 receptor immunoreactivity was expressed by lamina propria macrophages and smooth muscle cells. PPARα receptor immunoreactivity was expressed by blood vessels, smooth muscle cells, and glial cells of the myenteric plexus. Cannabinoid receptors showed a wide distribution in the gastrointestinal tract of the dog. Since cannabinoid receptors have a protective role in inflammatory bowel disease, the present research provides an anatomical basis supporting the therapeutic use of cannabinoid receptor agonists in relieving motility disorders and visceral hypersensitivity in canine acute or chronic enteropathies.     

New Vistas on the Recruiting of Ferrous Iron and Dioxygen by Ferritins: A Case Study of the Escherichia coli 24‐mer Ferritin by All‐Atom Molecular Dynamics in Aqueous Medium

It is shown here that Fe²⁺ and O₂ ligands are displaced from the ferroxidase center of the C1 four‐helix bundle of E. coli 24‐mer ferritin under molecular dynamics (MD) aided by a randomly oriented external force applied to the ligand. Under these conditions, ligand egress toward the external aqueous medium occurs preferentially from the same four‐helix bundle, in the case of O₂, or other bundle, in the case of Fe². Viewing ligand egress from the protein as the microscopic reverse of ligand influx into the protein under unbiased MD, these findings challenge current views that preferential gates for recruitment of Fe²⁺ are 3‐fold channels with human ferritin, or the short path from the ferroxidase center to H93 with bacterial ferritins.     

Presence of nontryptophan fluorophores specifically bound to gamma 2-crystallin

alpha-, beta-, and gamma-crystallins have been purified from nonpathological lenses of calves. The pure proteins have been examined for nontryptophan fluorescence and fluorescent compounds have been found specifically bound to gamma 2-crystallin. The protein has been unfolded by 6 M guanidine hydrochloride (Gdn-HCl) and a separation of the fluorescent compounds has been obtained by gel chromatography in the presence of 6 M Gdn-HCl. The spectroscopic features (absorbance, fluorescence) of the protein returned to normal following removal of the chromophores. The low-molecular-weight separated fluorescent compounds have been fractionated and extracted from the Gdn-HCl solution by ethyl acetate. TLC chromatography has shown the presence of kynurenine, 3-OH-kynurenine, and free tryptophan. These data suggest that direct involvement of the intrinsic protein tryptophans in the photochemical processes leading to formation of fluorescent compounds has to be excluded. Free tryptophan and intrinsic metabolic factors are probably more relevant in determining the cataractous insult.     

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)]₄, we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG^BrGGT)]₄ and [d(TGGGG^BrT)]₄. The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)]₄ with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far.     

Novel peptidomimetics as BACE-1 inhibitors: Synthesis,molecular modeling,and biological studies

Aiming at identifying new scaffolds for BACE-1 inhibition devoid of the pharmacokinetic drawbacks of peptide-like structures, we investigated a series of novel peptidomimetics based on a 1,4-benzodiazepine (BDZ) core 1a–h and their seco-analogues 2a–d. We herein discuss synthesis, molecular modeling and in vitro studies which, starting from 1a, led to the seco-analogues (R)-2c and (S)-2d endowed with BACE-1 inhibition properties in the micromolar range both on the isolated enzyme and in cellular studies. These data can encourage to pursue these analogues as hits for the development of a new series of BACE-1 inhibitors active on whole-cells.     

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non‐hair cells and represents a model system for studying the control of cell‐fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell‐fate stabilization. Our work opens the door for future studies addressing SAB‐dependent functions of the cytoskeleton during root epidermal patterning.     

On the Quest of Dioxygen by Monomeric Sarcosine Oxidase. A Molecular Dynamics Investigation

It is reported here on random acceleration molecular dynamics (RAMD) simulations with the 2GF3 bacterial monomeric sarcosine oxidase (MSOX), O₂, and furoic acid in place of sarcosine, solvated by TIP3 H₂O in a periodic box. An external tiny force, acting randomly on O₂, accelerated its relocation, from the center of activation between residue K265 and the si face of the flavin ring of the flavin adenine dinucleotide cofactor, to the surrounding solvent. Only three of the four O₂ gates previously described for this system along a composite method technique were identified, while two more major O₂ gates were found. The RAMD simulations also revealed that the same gate can be reached by O₂ along different pathways, often involving traps for O₂. Both the residence time of O₂ in the traps, and the total trajectory time for O₂ getting to the solvent, could be evaluated. The new quick pathways discovered here suggest that O₂ exploits all nearby interstices created by the thermal fluctuations of the protein, not having necessarily to look for the permanent large channel used for uptake of the FADH cofactor. To this regard, MSOX resembles closely KijD3 N‐oxygenase. These observations solicit experimental substantiation, in a long term aim at discovering whether gates and pathways for the small gaseous ligands inside the proteins are under Darwinian functional evolution or merely stochastic control operates.     

Olfactory sensitivity to amino acids in the juvenile stages of the European eel Anguilla anguilla (L.)

Scanning electron micrograph observations of the olfactory mucosa from both unpigmented glass eel(GE)andpigmentedelvers(EL)of the European eel, Anguilla anguilla (L.), revealed the presence of various cell types; amongst these, the ciliated and microvillous ones are likely to possess a chcmosensory function. Recording of underwater electro-olfactograms (EOGs) showed that various amino acids (glycine, L-alanine, L-valine, L-leucine, L-asparagine, L-glutamine and L-methionine) are effective stimulants for the olfactory mucosa. Dose response curves of stimulus concentrations v. EOG amplitudesfit regression linesat both GE and EL stages. Leucine was more stimulatory at the GE than at the EL stage. The stimulatory effect of the other six amino acids tested was similar at both developmental stages. The possible role of olfactory sensitivity in animal behaviour at different developmental stages is discussed.