On the structure and synthesis of neuroprotectin D1, a novel anti-inflammatory compound of the docosahexaenoic acid family

Potato tuber lipoxygenase was shown to convert 17(S)-hydro(pero)xydocasahexaenoic acid in 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diHDHA] which was formed apparently through a double lipoxygenation mechanism. No traces of 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13E,15Z,19Z-enoic acid were found among the reaction products. It is very likely that a described earlier "neuroprotectin D1" [or "10,17(S)docosatriene"], a novel and potent anti-inflammatory compound derived from docosahexaenoic acid, was, in fact, 10,17(S)-dihydroxydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid formed through a double lipoxygenation mechanism instead of a previously thought epoxidation/isomerization mechanism.     

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.     

Regulating leukotriene synthesis: the role of nuclear 5-lipoxygenase

Leukotrienes are lipid messengers involved in autocrine and paracrine cellular signaling. They are synthesized from arachidonic acid by the 5-lipoxygenase pathway. Current models of this enzymatic pathway recognize that a key step in initiating leukotriene synthesis is the calcium-mediated movement of enzymes, including 5-lipoxygenase, to intracellular membranes. However, 5-lipoxygenase can be imported into or exported from the nucleus before calcium activation. As a result, its subcellular localization will affect its ability to be activated by calcium, as well as the membrane to which it binds and its interaction with other enzymes. This commentary focuses on the role of 5-lipoxygenase compartmentation in determining its regulation and, ultimately, leukotriene synthesis.     

EMSA法分析STAT5信号分子与周期蛋白cyclin D1的相互作用

利用蛋白凝胶电脉迁移率变动分析法（EMSA）分析STAT5信号分子与周期蛋白cyclinD1的相互作用。探讨位于cyclinD1启动子区的－248到－220之间,该区域包含SATAT5与cyclinD1的结合序列TTN5AA。STAT5与cyclinD1共形成a、b两条蛋白滞后带,当用点突变的探针作用时,仅剩下滞后带a,因此滞后带b可能为STAT5与cyclinD1的特异性结合条带。     

Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A(4) (LTA(4)) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC(50) values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5(S)-hydroperoxyeicosatetraenoic acid (5-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX.     

Synthesis,anti-inflammatory,analgesic, 5-lipoxygenase (5-LOX) inhibition activities,and molecular docking study of 7-substituted coumarin derivatives

In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type of inhibition with 5-LOX enzyme. Presence of OCH₃ group in 35 and Cl in 33 at C6-position of benzothiazole ring were found very important substitutions for potent activity.     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

Glucagon or cyclic AMP-stimulated synthesis of 5-phosphoribosyl 1-pyrophosphate in isolated hepatocytes and inhibition by antimicrotubular drugs.

Glucagon increased the level of 5-phosphoribosyl 1-pyrophosphate ($\text{PP}$Rib$\text{P}$) in isolated rat hepatocytes; a relatively high concentration of cyclic AMP could replace glucagon. In the presence of glucagon, the rate of incorporation of respective radioactive precursors into purine, pyrimidine, and oxidized pyridine nucleotides was accelerated, indicating that glucagon stimulates the synthesis of $\text{PP}$Rib$\text{P}$. Addition of 10^?6 M colchicine, vinblastin, or podophyllotoxin abolished the glucagon or cyclic AMP-induced increase in the $\text{PP}$Rib$\text{P}$ level. Colchicine did not affect accumulation of cyclic AMP induced by glucagon. These results suggest the involvement of tubulin or microtubules in the signal transfer from cyclic AMP to stimulated synthesis of $\text{PP}$Rib$\text{P}$.     

5-Hydroxyeicosatetraenoic acid and human parturition

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE₂, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p < 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ± 1.5, ASA treatment alone 5.17 ± 0.20, IL-1β alone 6.39 ± 2.1, and ASA + IL-1β 8.95 ± 1.2 (mean ± SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor.     

Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

A 1 846 bp cDNA is isolated from a human tonsil cell λ gt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.     

Solution structure of LC5, the CCR5‐ derived peptide for HIV‐1 inhibition

The synthetic peptide fragment (LC5: LRCRNEKKRHRAVRLIFTI) inhibits human immunodeficiency virus type 1 (HIV‐1) infection of MT‐4 cells. In this study, the solution structure of LC5 in SDS micelles was elucidated by using the standard ¹H two‐dimensional NMR spectroscopic method along with circular dichroism and fluorescence quenching. The peptide adopts a helical structure in the C‐terminal region (residues 13–16), whereas the N‐terminal part remains unstructured. The importance of Phe17 in maintaining the structure of LC5 was demonstrated by replacing Phe17 with Ala, which resulted in the dramatic conformational change of LC5. The solution structure of LC5 elucidated in the present work provides a basis for further study of the mechanism of the inhibition of HIV‐1 infection. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic variation at minisatellite loci D1S7, D4S139, D5S110 and D17S79 among three population groups of eastern India

Genetic variation at four minisatellite loci D1S7, D4S139, D5S110 and D17S79 in three predominant population groups of eastern India, namely Brahmin, Kayastha and Garo, are reported in this study. The Brahmin and Kayastha are of Indo-Caucasoid origin while the Garo community represents the Indo-Mongoloid ethnic group. The methodology employed comprised generation of HaeIII-restricted fragments of isolated DNA, Southern blotting, and hybridization using chemiluminescent probes MS1, pH30, LH1 and V1 for the four loci. All four loci were highly polymorphic in the population groups. Heterozygosity values for the four loci ranged between 0.68 and 0.95. Neither departure from Hardy-Weinberg expectations nor evidence of any association across alleles among the selected loci was observed. The gene differentiation value among the loci is moderate (G_ST = 0.027). A neighbour-joining tree constructed on the basis of the generated data shows very low genetic distance between the Brahmin and Kayastha communities in relation to the Garo. Our results based on genetic distance analysis are consistent with results of earlier studies based on serological markers and linguistic as well as morphological affiliations of these populations and their Indo-Caucasoid and Indo-Mongoloid origin. The minisatellite loci studied here were found to be not only useful in showing significant genetic variation between the populations but also to be suitable for human identity testing among eastern Indian populations.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

A new Cu/Zn carboxylato-bridged 1D polymer: Direct synthesis, X-ray structure and magnetic properties

A new complex [Cu₂Zn(O₂CMe)₆(NH₃)₂]_n was isolated as an unexpected product in an open-air synthesis of a mixed-metal compound using zero-valent copper, zinc oxide and ammonium acetate in the presence of 2-diethylaminoethanol in acetonitrile solution. Its structure consists of Cu₂(O₂CMe)₄ units situated on crystallographic inversion centres and Zn(NH₃)₂(O₂CMe)₂ units on crystallographic 2-fold axes. One O atom of each of the O₂CMe groups is attached via Zn bridges to the Cu atoms of the dimeric Cu₂(O₂CMe)₄ unit forming a 1D polymer in the bc direction. The polymer is not linear, with the dihedral angles between successive Cu-Cu vectors being 48.9°. Within the Cu₂(O₂CMe)₄ units the Cu···Cu distance is 2.675(2) Å and the angles between the O₂CMe planes are 88.8(5)°. The magnetic properties have been analyzed using the Hamiltonian ? = J?₁?₂ with J = 286 cm⁻¹ and g = 2.13. High-field EPR spectra showed both the exchange-coupled copper pair and non-interacting copper(II) ions. The presence of the latter species was explained as an effect of zinc atoms occupying a fraction of the copper sites.     

Functional variants of the human 5-lipoxygenase gene and their genetic diagnosis

Variants in the 5-lipoxygenase (ALOX5) gene are first-line candidate causes for interindividual differences in diseases where leukotrienes play a key role, e.g., inflammatory and immune diseases, atherosclerosis, asthma or the acute respiratory distress syndrome (ARDS). We developed and validated Pyrosequencing™ screening assays for single nucleotide polymorphism (dbSNP-IDs rs4986832, rs4987105, rs2115819, rs3740107, rs1565096, rs2291427, rs10571382, rs2242334, rs2229136, rs3802548), and a capillary electrophoresis assay for the ALOX5 Sp1/Egr1 promoter tandem repeat polymorphism. This selection spans the whole ALOX5 gene range and includes all variants with reported functional associations. A gene structure analysis in DNAs from 187 healthy unrelated Caucasians revealed two haploblocks, one in the promoter and one spanning six SNPs from rs3740107G>A in intron 6 to rs2229136A>G in exon 13. The five-repeat genotype was the most frequent Sp1/Egr1 promoter tandem repeat variant (allelic frequency 84%). These assays and analyses provide a solid basis for future assessments of the genetic modulation of leukotriene production.     

Flavivirus RNA cap methyltransferase: structure,function, and inhibition

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5′ terminal cap 1 structure (m⁷GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m⁷GpppA → m⁷GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2′-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2′-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.