A potent and selective inhibitor targeting human and murine 12/15-LOX

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood–brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC₅₀ potency of 3.4 ± 0.5 μM against h12/15-LOX in vitro and an ex vivo IC₅₀ potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a K_ic of 1.0 ± 0.08 μM and a K_iu of 6.0 ± 3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC_{50 L. braziliensis} 30.47 ± 6.35 μM; IC_{50 L. amazonensis} 40.46 ± 14.21 μM; IC_{50 L. infantum} 65.93 ± 15.12 μM) and amastigotes (IC_{50 L. braziliensis} 68.75 ± 16.55 μM; IC_{50 L. amazonensis} 38.45 ± 12.05 μM; IC_{50 L. infantum} 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC₅₀ 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.     

Isatin based Schiff bases as inhibitors of α-glucosidase: Synthesis,characterization, in vitro evaluation and molecular docking studies

Isatin base Schiff bases (1–20) were synthesized, characterized by ¹H NMR and EI/MS and evaluated for α-glucosidase inhibitory potential. Out of these twenty (20) compounds only six analogs showed potent α-glucosidase inhibitory potential with IC₅₀ value ranging in between 2.2 ± 0.25 and 83.5 ± 1.0 μM when compared with the standard acarbose (IC₅₀ = 840 ± 1.73 μM). Among the series compound 2 having IC₅₀ value (18.3 ± 0.56 μM), 9 (83.5 ± 1.0 μM), 11 (3.3 ± 0.25 μM), 12 (2.2 ± 0.25 μM), 14 (11.8 ± 0.15 μM), and 20 (3.0 ± 0.15 μM) showed excellent inhibitory potential many fold better than the standard acarbose. The binding interactions of these active analogs were confirmed through molecular docking.     

Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis,biological evaluation and molecular modeling studies

A series of new biphenyl bis-sulfonamide derivatives 2a–3p were synthesized in good to excellent yield (76–98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC₅₀ 0.04 ± 0.0001 μM for AChE) and (IC₅₀ 0.85 ± 0.0001 μM for BChE), the IC₅₀ values of these compounds were ranged from 2.27 ± 0.01 to 123.11 ± 0.04 μM for AChE and 7.74 ± 0.07 to <400 μM for BuChE. Among the tested compounds, 3p was found to be the most potent against AChE (IC₅₀ 2.27 ± 0.01 μM), whereas 3g exhibited the highest inhibition for BChE (IC₅₀ 7.74 ± 0.07 μM). Structure–activity relationship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.     

Synthesis of 6-chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives: Antidiabetic,antioxidant, β-glucuronidase inhibiton and their molecular docking studies

6-Chloro-2-Aryl-1H-imidazo[4,5-b]pyridine derivatives 1–26 were synthesized and characterized by various spectroscopic techniques. All these derivatives were evaluated for their antiglycation, antioxidant and β-glucuronidase potential followed their docking studies. In antiglycation assay, compound 2 (IC₅₀ = 240.10 ± 2.50 μM) and 4 (IC₅₀ = 240.30 ± 2.90 μM) was found to be most active compound of this series, while compounds 3 (IC₅₀ = 260.10 ± 2.50 μM), 6 (IC₅₀ = 290.60 ± 3.60 μM), 13 (IC₅₀ = 288.20 ± 3.00 μM) and 26 (IC₅₀ = 292.10 ± 3.20 μM) also showed better activities than the standard rutin (IC₅₀ = 294.50 ± 1.50 μM). In antioxidant assay, compound 1 (IC₅₀ = 69.45 ± 0.25 μM), 2 (IC₅₀ = 58.10 ± 2.50 μM), 3 (IC₅₀ = 74.25 ± 1.10 μM), and 4 (IC₅₀ = 72.50 ± 3.30 μM) showed good activities. In β-glucuronidase activity, compounds 3 (IC₅₀ = 29.25 ± 0.50 μM), compound 1 (IC₅₀ = 30.10 ± 0.60 μM) and compound 4 (IC₅₀ = 46.10 ± 1.10 μM) showed a significant activity as compared to than standard D-Saccharic acid 1,4-lactonec (IC₅₀ = 48.50 ± 1.25 μM) and their interaction with the enzyme was confirm by docking studies.     

Identification of novel acetylcholinesterase inhibitors: Indolopyrazoline derivatives and molecular docking studies

The synthesis of novel indolopyrazoline derivatives (P1-P4 and Q1-Q4) has been characterized and evaluated as potential anti-Alzheimer agents through in vitro Acetylcholinesterase (AChE) inhibition and radical scavenging activity (antioxidant) studies. Specifically, Q3 shows AChE inhibition (IC₅₀: 0.68 ± 0.13 μM) with strong DPPH and ABTS radical scavenging activity (IC₅₀: 13.77 ± 0.25 μM and IC₅₀: 12.59 ± 0.21 μM), respectively. While P3 exhibited as the second most potent compound with AChE inhibition (IC₅₀: 0.74 ± 0.09 μM) and with DPPH and ABTS radical scavenging activity (IC₅₀: 13.52 ± 0.62 μM and IC₅₀: 13.13 ± 0.85 μM), respectively. Finally, molecular docking studies provided prospective evidence to identify key interactions between the active inhibitors and the AChE that furthermore led us to the identification of plausible binding mode of novel indolopyrazoline derivatives. Additionally, in-silico ADME prediction using QikProp shows that these derivatives fulfilled all the properties of CNS acting drugs. This study confirms the first time reporting of indolopyrazoline derivatives as potential anti-Alzheimer agents.     

Synthesis and biological evaluation of indole derivatives as α-amylase inhibitor

A series of twenty indole hydrazone analogs (1–21) were synthesized, characterized by different spectroscopic techniques such as ¹H NMR and EI-MS, and screened for α-amylase inhibitory activity. All analogs showed a variable degree of α-amylase inhibition with IC₅₀ values ranging between 1.66 and 2.65 μM. Nine compounds that are 1 (2.23 ± 0.01 μM), 8 (2.44 ± 0.12 μM), 10 (1.92 ± 0.12 μM), 12 (2.49 ± 0.17 μM), 13 (1.66 ± 0.09 μM), 17 (2.25 ± 0.1 μM), 18 (1.87 ± 0.25 μM), 20 (1.83 ± 0.63 μM), and 19 (1.97 ± 0.02 μM) showed potent α-amylase inhibition when compared with the standard acarbose (1.05 ± 0.29 μM). Other analogs showed good to moderate α-amylase inhibition. The structure activity relationship is mainly focusing on difference of substituents on phenyl part. Molecular docking studies were carried out to understand the binding interaction of the most active compounds.     

Synthesis,in vitro α-glucosidase inhibitory activity and molecular docking studies of new thiazole derivatives

Current study based on the synthesis of new thiazole derivatives via “one pot” multicomponent reaction, evaluation of their in vitro α-glucosidase inhibitory activities, and in silico studies. All synthetic compounds were fully characterized by ¹H NMR, ¹³C NMR and EIMS. CHN analysis was also performed. These newly synthesized compounds showed activities in the range of IC₅₀ = 9.06 ± 0.10–82.50 ± 1.70 μM as compared to standard acarbose (IC₅₀ = 38.25 ± 0.12 μM). It is worth mentioning that most of the compounds such as 1 (IC₅₀ = 23.60 ± 0.39 μM), 2 (IC₅₀ = 22.70 ± 0.60 μM), 3 (IC₅₀ = 22.40 ± 0.32 μM), 4 (IC₅₀ = 26.5 ± 0.40 μM), 6 (IC₅₀ = 34.60 ± 0.60 μM), 7 (IC₅₀ = 26.20 ± 0.43 μM), 8 (IC₅₀ = 14.06 ± 0.18 μM), 9 (IC₅₀ = 17.60 ± 0.28 μM), 10 (IC₅₀ = 27.16 ± 0.41 μM), 11 (IC₅₀ = 19.16 ± 0.19 μM), 12 (IC₅₀ = 9.06 ± 0.10 μM), 13 (IC₅₀ = 12.80 ± 0.21 μM), 14 (IC₅₀ = 11.94 ± 0.18 μM), 15 (IC₅₀ = 16.90 ± 0.20 μM), 16 (IC₅₀ = 12.60 ± 0.14 μM), 17 (IC₅₀ = 16.30 ± 0.29 μM), and 18 (IC₅₀ = 32.60 ± 0.61 μM) exhibited potent inhibitory potential. Molecular docking study was performed in order to understand the molecular interactions between the molecule and enzyme. Newly identified α-glucosidase inhibitors except few were found to be completely non-toxic.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and solid-state cultures and recovery of hydrophobin proteins

The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-state (SSC) cultures. The maximum CO₂ production rate in SmC (V_max = 11.7 mg CO₂ L_g⁻¹ day⁻¹) was three times lower than in SSC (V_max = 40.4 mg CO₂ L_g⁻¹ day⁻¹). The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein⁻¹, but in SmC, this protein was not detected. The PLHYD showed a critical micelle concentration of 0.45 mg mL⁻¹. In addition, the PLHYD modified the hydrophobicity of Teflon from 130.1 ± 2° to 47 ± 2°, forming porous structures with some filaments <1 μm and globular aggregates <0.25 μm diameter. The interfacial studies of this PLHYD could be the basis for the use of the protein to modify surfaces and to stabilize compounds in emulsions.     

Kinetic characterization of human phosphopantothenoylcysteine synthetase

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and l-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10 mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 μM, 57 μM, and 16 μM for CTP, PPA, and cysteine, respectively, with a k_cat of 0.53 ± 0.01 s^? 1 for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 μM, 13 μM, and 14 μM for ATP, PPA, and cysteine, respectively, with a k_cat of 0.56 s^? 1 for the reaction. Oxygen transfer studies found that ¹⁸O from [carboxyl-¹⁸O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.     

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b₅₆₂ by 71%, in an [O₂] independent manner. Hyperbolic increases in O₂^•- (up to 1.3±0.1 nmol/min. mg protein) and H₂O₂ (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O₂^•-/H₂O₂ ratio was 1.98 in accordance with the stoichiometry of the O₂^•- disproportionation. Moreover, H₂O₂ production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N₂ atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH^•]_ss which, in turn, leads to an increase in O₂^•- and H₂O₂ mitochondrial production rates.     

Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.)

To test the hypothesis that impaired mitochondrial respiration limits cardiac performance at warm temperatures, and examine if any effect(s) are sex-related, the consequences of high temperature on cardiac mitochondrial oxidative function were examined in 10 °C acclimated, sexually immature, male and female Atlantic cod. Active (State 3) and uncoupled (States 2 and 4) respiration were measured in isolated ventricular mitochondria at 10, 16, 20, and 24 °C using saturating concentrations of malate and pyruvate, but at a submaximal (physiological) level of ADP (200 µM). In addition, citrate synthase (CS) activity was measured at these temperatures, and mitochondrial respiration and the efficiency of oxidative phosphorylation (P:O ratio) were determined at [ADP] ranging from 25–200 µM at 10 and 20 °C. Cardiac morphometrics and mitochondrial respiration at 10 °C, and the thermal sensitivity of CS activity (Q₁₀=1.51), were all similar between the sexes. State 3 respiration at 200 µM ADP increased gradually in mitochondria from females between 10 and 24 °C (Q₁₀=1.48), but plateaued in males above 16 °C, and this resulted in lower values in males vs. females at 20 and 24 °C. At 10 °C, State 4 was ~10% of State 3 values in both sexes [i.e. a respiratory control ratio (RCR) of ~10] and P:O ratios were approximately 1.5. Between 20 and 24 °C, State 4 increased more than State 3 (by ~70 vs. 14%, respectively), and this decreased RCR to ~7.5. The P:O ratio was not affected by temperature at 200 μM ADP. However, (1) the sensitivity of State 3 respiration to increasing [ADP] (from 25 to 200 μM) was reduced at 20 vs. 10 °C in both sexes (K_m values 105±7 vs. 68±10 μM, respectively); and (2) mitochondria from females had lower P:O values at 25 vs. 100 μM ADP at 20 °C, whereas males showed a similar effect at 10 °C but a much more pronounced effect at 20 °C (P:O 1.05 at 25 μM ADP vs. 1.78 at 100 μM ADP). In summary, our results demonstrate several sex-related differences in ventricular mitochondrial function in Atlantic cod, and suggest that myocardial oxidative function and possibly phosphorylation efficiency may be limited at temperatures of 20 °C or above, particularly in males. These observations could partially explain why cardiac function in Atlantic cod plateaus just below this species׳ critical thermal maximum (~22 °C) and may contribute to yet unidentified sex differences in thermal tolerance and swimming performance.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

Isolation and characterization of sesquiterpenes from Arecophila saccharicola YMJ96022401 with NO production inhibitory activity

Sesquiterpenes, arecoic acids A–F and arecolactone, were isolated from the ethyl acetate extracts of the fermented broth of Arecophila saccharicola YMJ96022401 along with two known analogues 1,7α,10α-trihydroxyeremophil-11(13)-en-12,8-olide and 1,10α,13-trihydroxyeremophil-7(11)-en-12,8-olide. Their structures were elucidated on the basis of spectroscopic data analyses. The inhibitory effects of all of these compounds on nitric oxide (NO) production in lipopolysaccharide (LPS, 200 μg/mL)-activated murine macrophage RAW264.7 cells were also evaluated. Among these compounds, 1,7α,10α-trihydroxyeremophil-11(13)-en-12,8-olide significantly inhibited NO production without any cytotoxicity, and its average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) were 85.7% ± 0.8% and 16.5 ± 1.0 μM, respectively. Arecolactone was the most potent, with the E_max at 12.5 μM and IC₅₀ being 94.7% ± 0.8% and 1.32 ± 0.1 μM, respectively, but displayed cytotoxicity at considerable higher concentrations than 25 μM. Analyses of Western blotting indicated that arecolactone (0.8–12.5 μM) inhibited induction of inducible NO synthase (iNOS) by LPS, which involved suppression of NF-κB activation and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) in activated RAW 264.7 cells. In addition, arecolactone concentration-dependently prevented the vascular hyporeactivity to phenylephrine induced by LPS (300 ng/mL) through iNOS pathway in isolated rat thoracic aortic rings. These results indicated that both of these naturally occurring iNOS inhibitors may provide a rationale for the potential anti-inflammatory effect of A. saccharicola YMJ96022401.     

Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of Haemanthus L. (Amaryllidaceae)

The alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two alkaloids constituted 88, 91 and 98% of the total alkaloid extract from each species respectively. GC–MS analysis revealed that H. coccineus and H. sanguineus had a relative abundance of coccinine (74 and 91% respectively) to montanine (14 and 7% respectively); whereas H. montanus had 20% coccinine and 71% montanine. The three extracts and two isolated alkaloids were evaluated for binding to the serotonin transporter protein (SERT) in vitro. Affinity to SERT was highest in H. coccineus (IC₅₀ = 2.0 ± 1.1 μg/ml) followed by H. montanus (IC₅₀ = 6.8 ± 1.0 μg/ml) and H. sanguineus (IC₅₀ = 28.7 ± 1.1 μg/ml). Montanine (IC₅₀ = 121.3 ± 3.6 μM or 36.56 ± 1.14 μg/ml; K_i = 66.01 μM) was more active than coccinine (IC₅₀ = 196.3 ± 3.8 μM or 59.15 ± 1.08 μg/ml; K_i = 106.8 μM), both of which were less active than the total alkaloid extracts of each species investigated. The possible synergistic effects of two coccinine/montanine mixtures (80:20 and 20:80) were investigated, however the mixtures gave similar activities as the pure compounds and did not show any increase in activity or activity similar to the total alkaloid extracts. Thus the considerably higher activity observed in the total alkaloid extracts is not correlated to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line. This indicates that P-gp efflux will not be limiting for blood–brain-barrier passage of the alkaloids.     

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1)

In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (K_f), effects that are overcome using an amphipathic quinone (CoQ₁) [Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψ_m) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O₂ consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ₁ was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O₂ consumption (1.5±0.1 nmol/min/mg protein), Δψ_m (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ₁ on rotenone-induced changes in ATP, O₂ consumption rate, Δψ_m, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ₁ were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM.     

Inhibition of mitochondrial membrane bound-glutathione transferase by mitochondrial permeability transition inhibitors including cyclosporin A

AimsEffect of mitochondrial permeability transition (MPT) inhibitors on mitochondrial membrane-bound glutathione transferase (mtMGST1) activity in rat liver was investigated in vitro.Main methodsWhen mitochondria were incubated with MPT inhibitors, mtMGST1 activity was decreased dose dependently and their 50% inhibition concentration (IC₅₀) were 1.2 μM (cyclosporin A; CsA), 31 μM (bongkrekic acid; BKA), 1.8 mM (ADP), and 3.2 mM (ATP). The decrease of mtMGST1 activity by the MPT inhibitors was not observed in the presence of detergent Triton X-100. On the contrary, mtMGST1 inhibition by GST inhibitors such as cibacron blue (IC₅₀, 4.2 μM) and S-hexylglutathione (IC₅₀, 480 μM) was not affected in the presence of detergent. Although mtMGST1 resides in both the inner (IMM) and outer mitochondrial membranes (OMM), only mtMGST1 in the IMM was inhibited by the MPT inhibitors in the absence of detergent. GST inhibitors decreased mtMGST1 activity both in the IMM and OMM regardless of the presence or absence of detergent. Cytosolic GSTs and microsomal MGST1 were not inhibited by the MPT inhibitors.Key findingsThese results indicate that mtMGST1 is inhibited by MPT inhibitors through membrane components, not directly by the inhibitors.SignificanceSince CsA binds to cyclophilin D (Cyp-D) in the mitochondrial matrix whereas BKA or ADP binds to adenine nucleotide translocator (ANT) in the IMM, it was suggested that mtMGST1 in the IMM interacts with Cyp-D/ANT and the binding of MPT inhibitors to Cyp-D or ANT causes their conformational change followed by an alteration of mtMGST1 conformation, resulting in decreasing mtMGST1 activity.