Isolation and characterization of four di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate (DBP)-degrading <Emphasis Type="Italic">Gordonia</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strains and cloning the 3,4-phthalate dioxygenase gene

Four aerobic bacterial strains capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy were isolated from river sediments. Based on the morphology, biochemical characterization, and 16S rRNA gene sequence analysis, they were identified as Gordonia sp. The optimal conditions for DBP degradation by these strains were found to be pH 7.0, 30°C, and stirring at 175 rpm. These four strains could degrade, respectively, 96, 98, 98, and 78% of DBP (400 mg l⁻¹) as well as dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-octyl phthalate (DOP), di-isooctyl phthalate (DIOP), and di-isononyl phthalate (DINP). Furthermore, partial sequences of the gene for 3,4-phthalate dioxygenase were obtained from all four strains. To our knowledge, this is the first time that the 3,4-phthalate dioxygenase gene has been successfully cloned from Gordonia sp.     

Co-Metabolic Biodegradation of DBP by Paenibacillus sp. S-3 and H-2

Two di-n-butyl phthalate (DBP)-degrading strains, designated as S-3 and H-2, were isolated from DBP-polluted soil and both identified as Paenibacillus sp. When DBP was provided as the sole carbon source, about 45.5 and 71.7 % of DBP (100 mg/L) were degraded by strain S-3 and H-2, respectively, after incubation for 48 h. However, DBP (100 mg/L) was degraded completely by co-culture of strain S-3 and H-2 after incubation for 60 h. Four phthalic acid (PA) esters could be utilized by co-metabolism in the study and the degradation rates followed the order of dimethyl phthalate > diethyl phthalate > DBP > dioctyl phthalate. The metabolic pathway of DBP was elucidated based on the results of metabolites identification and enzyme assays. For strain S-3, DBP was degraded into butyl hydrogen phthalate which was degraded to PA by carboxyesterase further. But PA could be not hydrolyzed further because strain S-3 lacked 3,4-phthalate dioxygenase. Different with S-3, strain H-2 could hydrolyze PA into 3,4-dihydroxy-PA by 3,4-phthalate dioxygenase. Then 3,4-dihydroxy-PA was converted to protocatechuate and benzoic acid. Finally, the aromatic ring was cleavage and mineralized to CO₂ and H₂O. Above all, co-metabolism could increase the activity of 3,4-phthalate dioxygenase and accelerated the degradation of DBP. This study highlights an important potential use of co-metabolic biodegradation for the in situ bioremediation of DBP and its metabolites-contaminated environment.     

Degradation of the endocrine-disrupting dimethyl phthalate carboxylic ester by Sphingomonas yanoikuyae DOS01 isolated from the South China Sea and the biochemical pathway

Bacteria capable of using dimethyl phthalate (DMP) as the sole carbon and energy source were isolated from the sediments collected at a depth of 1340 m from the South China Sea. Sphingomonas yanoikuyae DOS01, identified based on 16S rRNA gene sequence, utilized DMP from an initial level of 180 mg l^?1 to non-detectable in 35 h at 30 °C, the optical density (OD₆₀₀) values increased over the time of incubation. Degradation intermediate monomethyl phthalate (MMP) accumulated up to 21.3 mg l^?1 and then disappeared in the culture medium. When MMP or another intermediate phthalate (PA) was used as the sole substrate, this strain was only capable of degrading MMP, but not PA. Total organic carbon (TOC) analysis of the culture medium suggested that both DMP and MMP were mineralized, but not PA. This strain from the deep-ocean sediment transforms DMP to MMP using a common biochemical pathway for DMP as reported before. Further esterase activity assays indicated that the enzyme induced by MMP has higher affinity than that by DMP for the substrate p-nitrophenyl acetate. Our results indicated that complete degradation of DMP by this marine microorganism may involve a new biochemical pathway.     

Potential of Ipomoea aquatica cultivars in phytoremediation of soils contaminated with di-n-butyl phthalate

Five different genotypic cultivars of Ipomoea aquatica commonly grown in Southeast Asia were cultivated to investigate their accumulation variation of di-n-butyl phthalate (DBP) and their potential for phytoremediation of three soils contaminated with DBP (4.5, 10.3 and 22.5 mg kg^?1). The results indicated different cultivar tolerance to DBP. DBP concentration in the shoots of the cultivars and residual DBP concentration in the soil were proportional to initial DBP concentrations in the soil and significantly different with different genotypic cultivars, indicating that the removal of DBP is cultivar-specific. DBP removal in the soil with indigenous DBP was higher than that in freshly DBP-spiked soils. The cultivars of local white-skin I. aquatica (cultivar V₅) and Taiwan filiform-leaf I. aquatica (cultivar V₁) presented the highest phytoremediation potential in the soil containing indigenous DBP and in freshly DBP-spiked soil, respectively. The translocation factor (TF, DBP concentration ratio of the shoots to the roots) and bioconcentration factor (BCF, DBP concentration ratio of the plant to the soil) also significantly varied with different cultivars, and they did not follow distribution profiles correlated to DBP removal indicating that phytoextraction was not the dominant DBP removal mechanism. Finally, the potential ability of different cultivars of enhancing biodegradation varied widely.     

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC₅₀ 15 μM) and D3 for MhpB (IC₅₀ 110 μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1 mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.     

Efficient removal of toxic phthalate by immobilized serine-type aldehyde-tagged esterase G

Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.     

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg⁻¹ polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O₂ l⁻¹ d⁻¹ and SB1 oxidised biphenyl at 426±25 mg O₂ l⁻¹ d⁻¹ in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.     

Structure-dependent inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 activities by phthalates

Phthalates are diesters of phthalic acid and an alcohol moiety. Phthalates have been classified as endocrine disruptors and have a broad range of effects with unknown mechanisms. Some of the effects of phthalate are consistent with disruptions of normal glucocorticoid homeostasis, and in particular, with defective function of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). In the present study, we tested 12 phthalate diesters and four monoesters for the inhibition of human and rat kidney 11β-HSD2. We examined the modes of inhibition and looked for a relationship between the potency for inhibition and the chemical structures. Of the phthalate diesters we tested, dipropyl phthalate (DPrP) and di-n-butyl phthalate (DBP) significantly inhibited both human and rat 11β-HSD2 activities. The IC₅₀s were 85.59 μM for DPrP and 13.69 μM for DBP when calculated for rat 11β-HSD2. As diesters, 8 of the phthalates did not affect 11β-HSD2 enzyme activity. Compared to the diesters that were inhibitory, the 8 non-inhibitory phthalates, had either fewer carbons, that is 1 or 2 carbons in the alcohol moiety, or more carbons, 5–10, as a branched or unbranched chain in the alcohol moeity. However, phthalates could be inhibitors with six carbons in the alcohol moiety if the carbons were cyclized, as in dicyclohexyl phthalate (DCHP), which inhibited rat 11β-HSD2 with an IC₅₀ of 32.64 μM. Thus, whether a phthalate is an inhibitor may reflect the size and shape of the compound. Although the diesters are the compounds used in manufacturing and present as environmental contaminants, it is the monoester metabolites that are detected in human serum and urine. We showed that mono (2-ethylhexyl) phthalate (MEHP) significantly inhibited human (IC₅₀ = 110.8 ± 10.9) and rat (121.8 ± 8.5 μM) 11β-HSD2 activity even though its parent compound, di(2-ethylhexyl) phthalate (DEHP) did not. MEHP was a competitive inhibitor of 11β-HSD2 enzymatic activity. We conclude that phthalates of a certain size act as competitive inhibitors.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

Enzymatic synthesis of phenolic lipids in solvent-free medium using flaxseed oil and 3,4-dihydroxyphenyl acetic acid

The enzymatic synthesis of phenolic lipids (PLs) by transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA) was investigated in solvent-free medium (SFM), using Novozym 435 from Candida antarctica as the biocatalyst. The effects of selected reaction parameters, water activity (a_w), enzyme concentration and agitation speed, were studied and optimized. Increasing the a_w of the reaction mixture from 0.18 to 0.38 resulted in a significant increase in the bioconversion yield from 62 to 77%. APCI–MS analysis confirmed the formation of six 3,4-dihydroxyphenyl acetoylated lipids, which were monolinolenyl, dioleyl, dilinolenyl, linoleyl linolenyl, oleyl linolenyl and oleyl linoleyl dihydroxyphenyl acetates. The highest enzymatic activity (178 nmol of PLs/g solid enzyme/min) was obtained using 40 mg of solid enzyme (400 PLU)/mL at agitation speed 150 rpm. Using the optimized conditions, the phenolic lipids showed a high relative proportion of linolenic acid (C_18:3 n?3) that increased from 57% in the flaxseed oil to 75 and 64% in the produced phenolic mono- and diacylglycerols, respectively. In addition, the synthesized phenolic lipids demonstrated a 7.2-fold lower radical scavenging activity than that of DHPA but half that of α-tocopherol.     

Studies on depigmenting activities of dihydroxyl benzamide derivatives containing adamantane moiety

Six diphenolic compounds containing adamantane moiety were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. The inhibitory activity of 4-adamantyl resorcinol 1 was similar to that of 4-n-butyl resorcinol in both assays. However, dihydroxyl benzamide derivatives 6a–e showed different inhibitory patterns. All derivatives significantly suppressed the cellular melanin formation without tyrosinase inhibitory activities. These behaviors indicated that the introduction of amide bond changes the binding mode of dihydroxyl groups to tyrosinase. Among derivatives, 6d (3,4-dihydroxyl compound) and 6e (2,3-dihydroxyl compound) showed stronger inhibitory activities (IC₅₀ = 1.25 μM and 0.73 μM, respectively) as compared to 4-n-butyl resorcinol (IC₅₀ = 21.64 μM) and hydroquinone (IC₅₀ = 3.97 μM). This study showed that the position of dihydroxyl substituent at aromatic ring is important for the intercellular inhibition of melanin formation, and also amide linkage and adamantane moiety enhance the inhibition.     

Extraction of trace amounts of pioglitazone as an anti-diabetic drug with hollow fiber liquid phase microextraction and determination by high-performance liquid chromatography-ultraviolet detection in biological fluids

The applicability of hollow fiber liquid phase microextraction (HF-LPME) for extraction and preconcentration of trace amounts of pioglitazone (PGL) as an anti-diabetic drug in biological fluids, prior to determination by high-performance liquid chromatography (HPLC), was evaluated. In this technique, the target drug was extracted into di-n-hexyl ether immobilized in the wall pores of a porous hollow fiber from 10 mL of the aqueous sample (source phase, SP) with pH 8.0, and then back extracted into the receiving phase (RP) with pH 2.2 located in the lumen of the hollow fiber. The extraction occurred due to a pH gradient between the two sides of the hollow fiber. After extracting for a prescribed time, 24 μL of the RP solution was taken back into the syringe and injected directly into a HPLC instrument for quantification. The Taguchi orthogonal array (OAD) experimental design with an OA₁₆ (4⁵) matrix was employed to optimize the HF-LPME conditions. Different factors affecting the HF-LPME efficiency such as the nature of organic solvent used to impregnate the membrane, pH of the SP and RP, stirring speed, extraction time and ionic strength were studied and optimized. Under the optimum conditions (di-n-hexyl ether as membrane impregnation solvent, pHs of the SP and RP equal to 8.0 and 2.2, respectively, extraction time of 30 min, stirring speed of 500 rpm and 10% (w/v) NaCl for adjusting the ionic strength), preconcentration factor of 180, linear dynamic range (LDR) of 2.5–250 μg L^?1 with good correlation of determination (r² > 0.998) and limit of detection (LOD) of 1.0 μg L^?1 were obtained for the target drug. The percent relative intra-day and inter-day standard deviations (RSDs%) based on five replicate determinations were 4.7 and 15%, respectively. Once LPME was optimized, the performance of the proposed technique was evaluated for the determination of PGL in different types of biological fluids such as plasma and urine samples. The results showed that the proposed HF-LPME method could be successfully applied to determine trace amounts of PGL in biological samples.     

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu²⁺ was added when the optical density at 600 nm (OD₆₀₀) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD₆₀₀ was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.     

Identification of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT)

Potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors containing 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived ureas were identified using structure-based design techniques. The new compounds displayed improved aqueous solubilities, determined using a high-throughput solubility assessment, relative to previously disclosed urea and amide-containing NAMPT inhibitors. An optimized 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-derived compound exhibited potent anti-NAMPT activity (18; BC NAMPT IC₅₀ = 11 nM; PC-3 antiproliferative IC₅₀ = 36 nM), satisfactory mouse PK properties, and was efficacious in a PC-3 mouse xenograft model. The crystal structure of another optimized compound (29; NAMPT IC₅₀ = 10 nM; A2780 antiproliferative IC₅₀ = 7 nM) in complex with the NAMPT protein was also determined.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Microbial degradation kinetics of solid alkane dissolved in nondegradable oil phase

An Acinetobacter species was isolated and found to be able to grow on crude oil n-alkanes and solid alkanes at room temperature as the sole carbon source. The growth of the isolate on n-heneicosane dissolved in non-biodegradable pristane has been studied. A kinetic model of the growth of microorganism on the hydrophobic substrate dissolved in non-biodegradable oil droplet assuming direct contact of cell with oil droplet was developed and validated with a model system of crude oil biodegradation. The model was focused on the substrate transport to the cell being contact with the surface of droplet. The high value of saturation constant of n-heneicosane, K_s = 0.086 kg m⁻³, and the maximum specific growth rate, μ_m = 0.60 h⁻¹, were obtained. The transport limitation was considered and estimated. The high value of attached cell fraction was reasonable to explain the observed growth rate by the direct contact model and varied with time till it reached a plateau at the stationary growth phase. By considering the direct contact of the cells with the surface of pristane and the transport of n-heneicosane to the cell, the degradation of hydrophobic substrate in the oil phase could be elucidated.     

Novel insights into cyclooxygenases,linoleate diol synthases,and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O₂. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n ? 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n ? 6 and n ? 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C₂₀ and C₁₈ fatty acids in the following order of rates: 20:2n ? 6 > 20:1n ? 6 > 20:3n ? 9 > 20:1n ? 9 and 18:3n ? 3 ≥ 18:2n ? 6 > 18:1n ? 6. 18:2n ? 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n ? 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n ? 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-²H]18:2n ? 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n ? 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-²H₂]- and [11S-²H]18:2n ? 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n ? 6 by COX-1 and 18:1n ? 9 by 8R-DOX were both accompanied by large D-KIE (> 20).     

Synthesis and monoamine transporter affinity of 3α-arylmethoxy-3β-arylnortropanes

A series of 3-arylnortrop-2-enes and 3α-arylmethoxy-3β-arylnortropanes were synthesized and evaluated for binding affinity at monoamine transporters. The 3-(3,4-dichlorophenyl)nortrop-2-ene (6e) exhibited high affinity for the SERT (K_i = 0.3 nM). The 3α-arylmethoxy-3β-arylnortropanes were generally SERT selective with the 3α-(3.4-dichlorophenylmethoxy)-3βphenylnortrop-2-ene (7c) possessing subnanomolar potency (K_i = 0.061 nM). However, 3α-(3,4-dichlorophenylmethoxy)-3β-phenylnortrop-2-ene (7b) exhibited high affinity at all three transporters [(DAT K_i = 22 nM), (SERT K_i = 6 nM) and (NET K_i = 101 nM)].     

Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC₅₀ (μM) DYRK1A = 11; CDK5 = 0.41; GSK-3 = 1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer’s disease.