Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2?±?0.4, 2.0?±?0.3, 1.6?±?0.2?µmol/mg/h/mM; p?CN1 (rCN1) efficiency (5.2?±?0.2?µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1^C102S) and 229 (Mut2^C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.     

Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags

To identify new vaccine candidates, Eimeria tenella expressed sequence tags (ESTs) from public databases were analysed for secretory molecules with an especially developed automated in silico strategy termed DNAsignalP. A total of 12,187 ESTs were clustered into 2881 contigs followed by a blastx search, which resulted in a significant number of E. tenella contigs with homologies to entries in public databases. Amino acid sequences of appropriate homologous proteins were analysed for the occurrence of an N-terminal signal sequence using the algorithm signalP. The resulting list of 84 entries comprised 51 contigs whose deduced proteins showed homologies to proteins of apicomplexan parasites. Based on function or localisation, we selected candidate proteins classified as (i) secreted proteins of Apicomplexa parasites, (ii) secreted enzymes, and (iii) transport and signalling proteins. To verify our strategy experimentally, we used a functional complementation system in yeast. For five selected candidate proteins we found that these were indeed secreted. Our approach thus represents an efficient method to identify secretory and surface proteins out of EST databases.     

Mechanisms of cholangiocyte responses to injury

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

The reaction of cytochrome c with [Fe(EDTA)(H2O)]

The interaction of horse ferricytochrome c with the reagents [Fe(EDTA)(H₂O)]⁻ and [Cr(CN)₆]³⁻ were studied at pH 7 and 25°C by ¹H-NMR spectroscopy. Two binding regions near to the heme crevice of cytochrome c were identified. Both regions bound both reagents but they exhibited different selectivities.

The relevance of this finding to the electron-transfer function of cytochrome c is discussed.     

Mechanisms of trovafloxacin hepatotoxicity: studies of a model cyclopropylamine-containing system

The mechanism for the hepatotoxicity of trovafloxacin remains unresolved. Trovafloxacin contains a cyclopropylamine moiety which has a potential to be oxidized to reactive intermediate(s) although other putative elements may exist. In this study, a drug model of trovafloxacin containing the cyclopropylamine substructure was synthesized. Chemical oxidation of the drug model by K₃Fe(CN)₆ and NaClO revealed that both oxidants oxidize this drug model to a reactive ,β-unsaturated aldehyde, 11. The structure of 11 was fully elucidated by LC/MS/MS and NMR analysis. These results suggested that P450s with heme-iron center and myeloperoxidase generating hypochlorous acid in the presence of chloride ion are capable of bioactivating the cyclopropylamine moiety of trovafloxacin. This deleterious metabolism may lead to eventual hepatotoxicity.     

Continuous soil carbon storage of old permanent pastures in Amazonia

Amazonian forests continuously accumulate carbon (C) in biomass and in soil, representing a carbon sink of 0.42–0.65 GtC yr^?1. In recent decades, more than 15% of Amazonian forests have been converted into pastures, resulting in net C emissions (~200 tC ha^?1) due to biomass burning and litter mineralization in the first years after deforestation. However, little is known about the capacity of tropical pastures to restore a C sink. Our study shows in French Amazonia that the C storage observed in native forest can be partly restored in old (≥24 year) tropical pastures managed with a low stocking rate (±1 LSU ha^?1) and without the use of fire since their establishment. A unique combination of a large chronosequence study and eddy covariance measurements showed that pastures stored between ?1.27 ± 0.37 and ?5.31 ± 2.08 tC ha^?1 yr^?1 while the nearby native forest stored ?3.31 ± 0.44 tC ha^?1 yr^?1. This carbon is mainly sequestered in the humus of deep soil layers (20–100 cm), whereas no C storage was observed in the 0‐ to 20‐cm layer. C storage in C4 tropical pasture is associated with the installation and development of C3 species, which increase either the input of N to the ecosystem or the C:N ratio of soil organic matter. Efforts to curb deforestation remain an obvious priority to preserve forest C stocks and biodiversity. However, our results show that if sustainable management is applied in tropical pastures coming from deforestation (avoiding fires and overgrazing, using a grazing rotation plan and a mixture of C3 and C4 species), they can ensure a continuous C storage, thereby adding to the current C sink of Amazonian forests.     

An infrared spectroscopic based method to measure membrane permeance in liposomes

A new method for studying membrane permeance in liposomes is described. The method uses liposomes fabricated to contain IR probe molecules with CN moieties in combination with attenuated total reflection—Fourier transform infrared (ATR-FTIR) spectroscopy. The liposomes are adsorbed on a TiO₂ coated ATR crystal and remain intact to flowing aqueous solutions. A change in permeance is determined by monitoring the time dependent decrease in the intensity of a band due to CN groups. It is shown that the transport of the probe molecule depends on the size of the probe molecule and the structure of the liposome membrane. A much clearer molecular understanding of membrane permeance is obtained when the information derived from transport of the probe molecules is combined with the membrane packing arrangement determined from the infrared bands due to the lipids.     

Alpha-2 receptors in the gastrointestinal system: a new therapeutic approach

Alpha-2 receptor activation mediates the inhibition of a number of gastrointestinal functions including gastric and intestinal secretions. Alpha-2 receptors are located in the brain and presynaptically on cholinergic nerve terminals; activation of either inhibits vagus nerve activity. Intestinal secretions are inhibited by postsynaptic alpha-2 receptors located on intestinal epithelial cells. Agents which selectively activate alpha-2 receptors in the gut may therefore be beneficial in treating gastric ulcers and diarrheal states. Two such agents which activate alpha-2 receptors in the gut are WHR-1370A [1-n-butoxy-3-(2,6-dimethylphenylcarbamoyl) guanidine hydrochloride] and lidamidine. WHR-1370A is a potent gastric antisecretory and antiulcer agent which inhibits the release of acetylcholine from the vagus nerve. WHR-1370A's activity is blocked by yohimbine. Lidamidine is a clinically effective antidiarrheal agent. Lidamidine's response is partially inhibited by yohimbine in animal diarrheal models. Alpha-2 agonists represent a new class of drugs which have a promising future in the treatment of gastrointestinal disorders.     

Antifungal effect and pore-forming action of lactoferricin B like peptide derived from centipede Scolopendra subspinipes mutilans

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74 nm and 1.4 nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.     

1D chain coordination assembly of [Mn4(hmp)6(NO3)2] single-molecule magnets linked by the photochromic [FeNO(CN)5] precursor

The first heterometallic chain cluster {[Mn₄(hmp)₆(NO₃)₂FeNO(CN)₅·4CH₃CN}_n (1) based on the [Mn₄(hmp)₆] SMM has been synthesized. 1 has one-dimensional chain structure: the [Mn₄(hmp)₆] units are linked via CN-groups of nitroprusside anions. Its magnetic and relaxation properties and low temperature IR spectra under light irradiation have been investigated. The ferromagnetic exchange constants have been calculated.