Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

miR-145-5p attenuates inflammatory response and apoptosis in myocardial ischemia-reperfusion injury by inhibiting (NADPH) oxidase homolog 1

Myocardial ischemia-reperfusion (I/R) injury is a common complication following reperfusion therapy that involves a series of immune or apoptotic reactions. Studies have revealed the potential roles of miRNAs in I/R injury. Herein, we established a myocardial I/R model in rats and a hypoxia/reoxygenation (H/R) model in H9c2 cells and investigated the effect of miR-145-5p on myocardial I/R injury. After 3 h or 24 h of reperfusion, left ventricular end-systolic pressure (LVESP), ejection fraction (EF), and fractional shortening (FS) were obviously decreased, and left ventricular end-diastolic pressure (LVEDP) was increased. Meanwhile, I/R induced an increase in myocardial infarction area. Moreover, a decrease in miR-145-5p and increase in (NADPH) oxidase homolog 1 (NOH-1) were observed following I/R injury. With this in mind, we performed a luciferase reporter assay and demonstrated that miR-145-5p directly bound to NOH-1 3’ untranslated region (UTR). Furthermore, miR-145-5p mimics decreased the levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-6 via oxygen and glucose deprivation/reperfusion (OGD/R) stimulation. Upregulation of miR-145-5p increased cell viability and reduced apoptosis accompanied by downregulation of Bax, cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP) and upregulation of Bcl2. In addition, miR-145-5p overexpression increased superoxide dismutase (SOD) activity and reduced reactive oxygen species (ROS) and malondialdehyde (MDA) content under OGD/R stress. Notably, NOH-1 could significantly abrogate the above effects, suggesting that it is involved in miR-145-5p-regulated I/R injury. In summary, our findings indicated that miR-145-5p/NOH-1 has a protective effect on myocardial I/R injury by inhibiting the inflammatory response and apoptosis.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Acute in vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone does not alter base excision repair activities in murine lung and liver

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen found in unburned tobacco and tobacco smoke, and is believed to play an important role in human tobacco-induced cancers. In previous studies, NNK has been reported to induce oxidative DNA damage, and to alter DNA repair processes, effects that could contribute to pulmonary tumorigenesis in rodent models. The goal of this study was to determine the effects of NNK on levels of 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA oxidation, and activity of base excision repair (BER), which repairs oxidative DNA damage. Female A/J mice were treated with a tumorigenic dose of NNK (10 μmol) i.p. At 1, 2 and 24 h post treatment, there were no statistically significant differences in lung or liver 8-OHdG levels between control and NNK-treated mice (P > 0.05). Furthermore, NNK did not alter lung or liver BER activity compared to control at any time point (P > 0.05). In summary, acute treatment with a tumorigenic dose of NNK did not stimulate oxidative DNA damage or significantly alter BER activity, and these effects may not be major mechanisms of action of NNK in mouse models.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Activity improvement of a regioselective nitrilase from Acidovorax facilis and its application in the production of 1-(cyanocyclohexyl) acetic acid

A nitrilase gene from Acidovorax facilis ZJB09122 was cloned and expressed in Escherichia coli BL21 (DE3). To improve the activity of this nitrilase, a key amino acid Phe168 was selected and mutated by site-directed mutagenesis, based on the homology modeling and previously described “hot spot” mutation. After mutation and screening, a mutant (Mut-F168V) with higher activity and stability was obtained. The nitrilase activity of Mut-F168V to hydrolyze 1-cyanocyclohexylacetonitrile was 39.52-fold compared with wild type A. facilis nitrilase (Wt-Acf-Nit). The values of K_m and V_max of Mut-F168V were markedly decreased to 1.89-fold and increased to 50.34-fold as compared to Wt-Acf-Nit, respectively. The biotransformation study showed that 1.0 M of 1-cyanocyclohexylacetonitrile could be regioselectively hydrolyzed to 1-(cyanocyclohexyl) acetic acid with 90% yield. The yield of 1-(cyanocyclohexyl) acetic acid by Mut-F168V was 66.19-fold compared to Wt-Acf-Nit after 1 h at the concentration of 1.0 M 1-cyanocyclohexylacetonitrile as substrate. The 1-(cyanocyclohexyl) acetic acid was subsequently isolated and characterized. The mutant (Mut-F168V) appears promising for potential applications for the industrial production of 1-(cyanocyclohexyl) acetic acid.     

Computational validation of 3-ammonio-3-(4-oxido- 1H-imidazol-1-ium-5-yl) propane-1, 1-bis (olate) as a potent anti-tubercular drug against mt-MetAP

The advent of Multi Drug Resistant (MDR) strain of Mycobacterium tuberculosis (TB) necessitated search for new drug targets for the bacterium. It is reported that 3.3% of all new tuberculosis cases had multidrug resistance (MDR-TB) in 2009 and each year, about 0.44 million MDR-TB cases are estimated to emerge and 0.15 million people with MDR-TB die. Keeping such an alarming situation under consideration we wanted to design suitable anti tubercular molecules for new target using computational tools. In the work Methionine aminopeptidase (MetAP) of Mycobacterium tuberculosis was considered as target and three non-toxic phenolic=ketonic compounds were considered as ligands. Docking was done with Flex X and AutoDock 4.2 separately. Ten proven inhibitors of MetAP were collected from literature with their IC50 and were correlated using EasyQSAR to generate QSAR model. Activity of ligands in question was predicted from QSAR. Pharmacophore for each docking was generated using Ligandscout 3.0. Toxicity of the ligands in question was predicted on Mobyle@rpbs portal and Actelion property explorer. Molecular docking with target showed that of all three ligands, 3-ammonio-3-(4-oxido-1H-imidazol-1-ium-5-yl) propane-1, 1-bis (olate) has highest affinity (- 37.5096) and lowest IC50 (4.46 µM). We therefore, propose that -3-ammonio-3-(4-oxido-1H-imidazol-1-ium-5-yl) propane-1,1- bis(olate) as a potent MetAP inhibitor may be a new anti-tubercular drug particularly in the context of Multi Drug Resistant Tuberculosis (MDR-TB).     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Neuroprotective function of R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane, [R-(-)-BPAP], against apoptosis induced by N-methyl(R)salsolinol, an endogenous dopaminergic neurotoxin, in human dopaminergic neuroblastoma SH-SY5Y cells

R-(-)-1-(Benzofuran-2-yl)-2-propylaminopentane HCl [R-(-)-BPAP] is one of "catecholaminergic and serotonergic enhancers", which were proposed to improve symptoms through increase in impulse-evoked release of monoamine neurotransmitters for Parkinson's disease. It was reported that (-)-BPAP up-regulated the synthesis of neurotrophic factors in mouse astrocytes, suggesting the neuroprotective potency of (-)-BPAP. In this paper, the neuroprotective function of (-)-BPAP and the related compounds was examined against apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], a possible pathogenic toxin in Parkinson's disease, in human dopaminergic neuroblastoma SH-SY5Y cells. The anti-apoptotic activity was confirmed with some of (-)-BPAP analogues, and the mechanism was found to be due to the direct stabilization of mitochondrial membrane potential and the induction of anti-apoptotic Bcl-2. The studies on structure-activity relationship demonstrated that the potency to stabilize the mitochondrial membrane potential depended on the absolute stereo-chemical structure of BPAP derivatives. The compounds with dextrorotation prevented the mitochondrial permeability transition, whereas those with levorotation did not. The presence of a propargyl or propyl group at the amino residue of R-(-)-1-(benzofuran-2-yl)-2-propylamine increased potency to stabilize the membrane potential and prevent apoptosis. R-FPFS-1169 and R-FPFS-1180 had more potent to induce Bcl-2 and prevent apoptosis than the corresponding S-enantiomers. These results are discussed with the possible application of BPAP derivatives as neuroprotective agents in Parkinson's disease and other neurodegenerative disorders.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.     

Inhibition of aquaporin-1 expression by RNAi protects against aristolochic acid I-induced apoptosis in human proximal tubular epithelial (HK-2) cells

The aim of this study was to investigate the protective effect of inhibition of aquaporin-1 (AQP1) expression against aristolochic acid I (AA-I)-induced apoptosis. HK-2 cells impaired by AA-I were used in this study as the cell model of aristolochic acid nephropathy. Apoptosis was studied by different methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays, flow cytometry, and caspase 3 activity assays. We compared AA-I-mediated apoptosis in HK-2 cells with or without knockdown of AQP1 expression by RNA interference. MTT assays showed that AA-I inhibited the viability of HK-2 cells in a time- and concentration-dependent manner. Apoptosis was evidenced by the results of the Annexin V/propidium iodide assay and the occurrence of a sub-G1 peak in cell-cycle analysis. The activity of caspase 3 was found to have been increased by AA-I in a concentration-dependent manner. However, AQP1 RNA interference provided protection against injury in cells treated with AA-I (40 μM) for 24 h and attenuated the number of apoptotic cells. These results suggested that AQP1 plays an important role in AA-I-induced apoptosis and that inhibition of AQP1 expression may protect HK-2 cells from AA-I-induced apoptotic damage.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.     

Synthesis of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazines that have antibacterial activity and also inhibit inorganic pyrophosphatase

Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) synthetase inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity versus Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.     

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.     

The effect of water stress on 1-(malonylamino)cyclopropane-1-carboxylic acid concentration in plant tissues

Since the discovery of1-(malonylamino)cyclopropane-1-carboxylic acid (MACC)as a major metabolite of both endogenous andexogenously applied 1-aminocyclopropane-1-carboxylicacid (ACC), it has become evident that the formationof MACC from ACC can act to regulate ethyleneproduction in certain tissues. Hence it was suggestedthat MACC could serve as an indicator of water-stresshistory in plant tissues. The accurate quantificationof MACC in plant tissues is essential forunderstanding the role of MACC in the regulation ofethylene biosynthesis.Hoffman et al. [15] described a method for themeasurement of MACC in which MACC was hydrolysed byHCl to ACC, which was then assayed by chemicaloxidation to form ethylene. Attempts have been made byothers to raise monoclonal antibodies to MACC so thatan immunoassay could be developed in order to gain adeeper understanding of stress-induced ethyleneproduction but no further publications have beenforthcoming.Here a method employing GC-MS is compared with theindirect assay for MACC, which is based uponhydrolysis of MACC to ACC and conversion of ACC byhypochlorite reagent to ethylene which is subsequentlyquantified by GC.     

Synthesis,biological evaluation and in silico studies of 5-(3-methoxybenzylidene)thiazolidine-2,4-dione analogues as PTP1B inhibitors

PTP1B (protein tyrosine phosphatase 1B) dephosphorylates the insulin receptor substrate and thus acts as a negative regulator of the insulin and leptin signalling pathway. Recently, it has been considered as a new therapeutic target of intervention for the treatment of type2 diabetes. A series of aryl/alkylsulfonyloxy-5-(3-methoxybenzylidene)thiazolidine-2,4-dione derivatives were synthesized, screened in vitro for their PTP1B inhibitory activity and in vivo for anti-hyperglycaemic activity. Docking results further helped in understanding the nature of interactions governing the binding mode of ligands inside the active site of PTP1B. Among the synthesized compounds, 13 and 16 were found to be potent PTP1B inhibitors having IC₅₀ of 7.31 and 8.73 μM respectively. Significant lowering of blood glucose level was observed in some of the synthesized compounds in in vivo study.     

Role of sphingosine 1-phosphate and lysophosphatidic acid in fibrosis

This review highlights an emerging role for sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in many different types of fibrosis. Indeed, both LPA and S1P are involved in the multi-process pathogenesis of fibrosis, being implicated in promoting the well-established process of differentiation of fibroblasts to myofibroblasts and the more controversial epithelial–mesenchymal transition and homing of fibrocytes to fibrotic lesions. Therefore, targeting the production of these bioactive lysolipids or blocking their sites/mechanisms of action has therapeutic potential. Indeed, LPA receptor 1 (LPA₁) selective antagonists are currently being developed for the treatment of fibrosis of the lung as well as a neutralising anti-S1P antibody that is currently in Phase 1 clinical trials for treatment of age related macular degeneration. Thus, LPA- and S1P-directed therapeutics may not be too far from the clinic. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.