New dual ligands for the D2 and 5-HT1A receptors from the group of 1,8-naphthyl derivatives of LCAP

More than 300 million people are suffering from depression, one of the civilization diseases in the 21st century. Serotonin 5-HT_1AR and dopamine D₂R play an important role in the treatment and pathogenesis of depression. Moreover, in recent years, the efficacy of dual 5-HT_1A/D₂ receptors ligands has been demonstrated in the fight against depression. In this work the new bulky arylpiperazine derivatives (LCAP) were synthesized in microwave radiation field. The affinities for the selected serotonin (5-HT_1A,5-HT_2A,5-HT₆,5-HT₇) and dopamine (D₂) receptors have been evaluated in vitro. Compounds 5.3a, 5.4, 5.1c, 5.3d, 5.2a are promising dual 5-HT_1AR/D₂R ligands. The SAR analysis were additionally supported with molecular docking studies.     

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 μM and ?H of ?20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ?CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Ų. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.     

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.     

The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (^SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The ^SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the ^SG-ligands promoted the β-elimination of the AP site followed by the formation of a covalent bond with the β-eliminated fragment without UVA irradiation.     

Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.     

Inhibition of intestinal copper absorption by divalent cations and low-molecular-weight ligands in the rat

l-histidine (His) has been shown to enhance the inhibitory effect of zinc on intestinal copper absorption. This study was aimed at examining whether this effect of His was also extended to the interactions of other divalent cations: ferrous iron, tin, and cobalt, using an in vivo perfusion system in rats. Copper absorption and intestinal content of this element significantly decreased in the presence of 2 mM His and ferrous iron. Iron accumulation was greater when His was present than when omitted. A fivefold excess of tin inhibited copper absorption only when His was present. Citrate, at the same concentration as His, had no effect on copper absorption, but hepatic copper levels were increased, as compared to the absence of either His or citrate. Addition of 0.5 or 1.0 mM cobaltous salt plus His resulted in a sharp decrease in copper intestinal absorption, with an increase in intestinal tissue retention. These results confirm earlier findings with zinc and His, and suggest that a general phenomenon, either accelerating the removal of copper from the intestinal lumen or increasing, the retention of this element by the intestinal tissue, is a common feature of the interaction between cations of similar electronic configuration to copper and a high-affinity ligand, such as His.     

Electroaffinity in paracellular absorption of hydrophilic d-dipeptides by sparrow intestine

We previously demonstrated size selectivity in the absorption of nonelectrolyte hydrosoluble probes in birds, presumably by the paracellular pathway. Our goal in this study was to determine the charge selectivity in the absorption of hydrosoluble d-dipeptides, because there have been no studies of the electroaffinity of this absorption pathway in birds. For this purpose isosmotic solutions with two hydrophilic d-dipeptides: serine-lysine (positive at pH 7.4) and serine-aspartic (negative at pH 7.4) were gavaged into the stomach in nonanesthetized house sparrows (Passer domesticus), and injected into the pectoralis with a syringe in different trials. Fractional absorption was calculated as F = [AUC by gavage)]/[AUC by injection] (AUC = area under the curve of plasma probe concentration vs. time). Fractional absorption was significantly higher for the positively charged than negatively charged dipeptide (respectively, F=0.30±0.05 vs. F=0.17±0.03). These findings give the first evidence of cation selectivity by the paracellular route in the absorption of hydrosoluble solutes in the small intestine in birds.     

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.     

Supplementation of pancreatic digestive enzymes alters the composition of intestinal microbiota in mice

Although pancreatic enzyme replacement therapy (PERT) is effective in the alleviation of pancreatic exocrine insufficiency (PEI)-related symptoms in patients with chronic pancreatitis, its mechanism of action is poorly understood. Recent studies suggest that the intestinal microbiota is associated with the pathogenesis of chronic pancreatitis. Therefore, we hypothesized that PERT exerts its effect by modifying the intestinal microbiota in addition to its presumed role in promoting fat and protein absorption. To explore the mechanism of action of PERT, we analyzed the intestinal microbiotas of two groups of mice treated with either pancrelipase or tap water by using 16S rRNA amplicon sequencing. The results revealed that the bacterial compositions of the pancrelipase-treated mice were significantly different from those of the control samples. Akkermansia muciniphila, a key beneficial bacterium in the intestinal tract, showed a higher relative abundance in the pancrelipase-treated samples than in the control samples. Lactobacillus reuteri, a widely used probiotic bacterium known to relieve intestinal inflammation, also showed a higher relative abundance in the pancrelipase-treated samples. These results suggested that PERT induces the colonization of beneficial bacteria, thereby contributing to the attenuation of PEI-associated symptoms in addition to improvement of the nutritional state.     

Gas chromatographic method for detection of urinary sucralose: application to the assessment of intestinal permeability

We developed a capillary column gas chromatography (CCGC) method for the measurement of urinary sucralose (S) and three other sugar probes including, sucrose, lactulose (L) and mannitol (M) for use in in vivo studies of intestinal permeability. We compared the capillary method with a packed column gas chromatography (PCGC) method. We also investigated a possible role for sucralose as a probe for the measurement of whole gut permeability. Sample preparation was rapid and simple. The above four sugars were detected precisely, without interference. We measured intestinal permeability using 5- and 24-h urine collections in 14 healthy volunteers. The metabolism of sugars was evaluated by incubating the intestinal bacteria with an iso-osmolar mixture of mannitol, lactulose and sucralose at 37 degrees C for 19 h. Sugar concentrations and the pH of the mixture were monitored. The use of the CCGC method improved the detection of sucralose as compared to PCGC. The average coefficient of variation decreased from 15% to 4%. It also increased the sensitivity of detection by 200-2000-fold. The GC assay was linear between sucralose concentrations of 0.2 and 40 g/l (r=1.000). Intestinal bacteria metabolized lactulose and acidified the media but did not metabolize sucralose or mannitol. The new method for the measurement of urinary sucralose permits the simultaneous quantitation of sucrose, mannitol and lactulose, and is rapid, simple, sensitive, accurate and reproducible. Because neither S nor M is metabolized by intestinal bacteria, and because only a tiny fraction of either sugar is absorbed, this pair of sugar probes appears to be available for absorption throughout the GI tract. Thus, the 24-h urinary concentrations of S and M, or the urinary S/M ratio following an oral dose of a sugar mixture, might be good markers for whole gut permeability.     

Changes in the inhibitory responses to electrical field stimulation of intestinal smooth muscle from Trichinella spiralis infected rats

Functional motor changes and morphological alterations have been associated with intestinal inflammation. The aim of this work was to study functional motor changes in inflamed and non-inflamed intestinal segments of Trichinella spiralis infected rats. Thickness of muscle layers and cell infiltration during infection were also evaluated. Segments of rat jejunum and ileum were placed in organ bath and relaxations of the longitudinal muscle in response to electrical field stimulation (EFS) were recorded. During the post-infection (PI) period EFS-induced relaxations in ileum were decreased. Maximal decreases in relaxation were found on day 14-23 PI for ileum, whereas non significant changes were observed in jejunal samples throughout the experimental period. The sensitivity of the EFS-induced relaxations to the NO synthase inhibitor Nω-nitro-l-arginine (L-NNA) and to the soluble guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) was decreased on day 14 PI for jejunum, whereas in the ileum it lasted from day 14-23 PI. The sensitivity of EFS-induced relaxations to apamin (a small conductance calcium activated potassium channel blocker) disappeared between day 6-23 PI for both jejunum and ileum. In contrast, the sensitivity of the EFS-induced relaxations to the K⁺ channel blockers tetraethylamonium (TEA) and tetrapenthylammonium (TPEA) chloride was similar for healthy tissue and for tissue obtained form infected animals. Distribution and density of NADPH-diaphorase positive neurons was similar in tissue obtained form healthy and infected animals. In conclusion, intestinal inflammation induces functional and structural changes in both worm-free and worm-positive intestinal segments. Increased muscle thickness was similar for both inflamed and noninflamed segments but the most prominent functional changes i.e. a long-lasting decrease of EFS-induced relaxation was found in non-inflamed ileal segments.     

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.     

Insights into water accessible pathways and the inactivation mechanism of proton translocation by the membrane-embedded domain of V-type ATPases

V-type ATPases are multi-protein complexes, which acidify cellular compartments in eukaryotes. They pump protons against an ion gradient, driven by a mechano-chemical framework that exploits ATP hydrolysis as an energy source. This process drives the rotation of the so-called c-ring, a membrane embedded complex in the V_o-domain of the V-type ATPase, resulting in translocation of protons across the membrane. One way in which the enzyme is regulated is by disassembly and reassembly of the V₁-domain with the V_o-domain, which inactivates and reactivates the enzyme, respectively. Recently, structural data for the isolated V_o-domain from S. cerevisiae in an inactivated state were reported, suggesting the location of previously unobserved proton access pathways within the cytoplasmic and luminal compartments of the stator subunit a in V_o. However, the structural rationale for this inactivation remained unclear. In this study, the water accessibility pathway at the cytoplasmic side is confirmed, and novel insights into the role of the luminal channel with respect to the inactivation mechanism are obtained, using atomic-resolution molecular dynamics simulations. The results show that protonation of the key-glutamate, located in the c-ring of the V_o-domain, and facing the luminal compartment is preserved, when residing in the V₁-depleted state. Maintaining the protonation of this essential glutamate is necessary to lock the luminal channel in the inactive, solvent-free state. Based on these theoretical observations and previous experimental results, a model of the proton translocation mechanism in the V_o-domain from V-type ATPases is proposed.     

Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore’s disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.     

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25 ± 10 Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH₂ was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-_c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH₂ and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)₃ units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.     

Calculations of apparent ruminal synthesis and intestinal absorption of biotin in dairy cows as influenced by the extraction method

Abstract

Biotin is present in nature either free or as biocytin, which is only degraded under the action of a specific enzyme: biotinidase. This enzyme is not included in analytical assays generally used. A method for sample preparation using biotinidase was developed in our laboratory before analysis by ELISA. Three cows equipped with duodenal and ileal cannulae were used to compare the effects of methods of sample preparation on calculations of apparent ruminal synthesis and intestinal absorption of biotin. There was no apparent ruminal synthesis of biotin, no matter whether free or total biotin was measured (p = 0.84). Results also suggested that rumen microbes cannot utilize nor degrade biocytin present in the feed. Estimates of apparent intestinal absorption were influenced by the sample preparation method (p = 0.002). Analysis of free biotin caused an artefact, suggesting intestinal synthesis of this vitamin; whereas determination of total biotin concentrations showed that absorption was taking place in the small intestine.     

Adaptation of subpopulations of the Norway spruce needle endophyte Lophodermium piceae to the temperature regime

Lophodermium piceae represents the most common Norway spruce needle endophyte. The aim of this study was to find out whether subpopulations of L. piceae in climatically different environments (in which Norway spruce occurs natively) are adapted to local thermal conditions. L. piceae’s ability for thermal adaptation was investigated by determining growth rates of 163 isolates in vitro at four different temperatures: 2, 6, 20 and 25 °C. Isolates were obtained between 1995 and 2010 from apparently healthy needles sampled in Finland, Poland, Switzerland, Italy and southeastern Siberia. The sampling sites represent seven climatically distinct locations. Results were evaluated in relation to the age and geographic origin of the isolate, in addition to the highest and lowest average monthly temperature of the sampling location. We found a significant correlation between the growth rate and the age of the isolate at 25 °C. Variation in growth rates between subpopulations was low compared to within subpopulations. Only at 2 °C did statistically significant differences between the average growth rates of subpopulations emerge. These results suggest that L. piceae covers the whole distribution area of Norway spruce but that generally the thermal reaction norm of its subpopulations has not changed according to local temperature ranges, despite high contrast in thermal conditions across this vast area. Therefore, it would appear that the thermal environment is not a crucial factor in assessing the fitness of this fungal species within the native range of Norway spruce.