Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of this study was to investigate the safety and efficacy of alum formulated glutamic acid decarboxylase GAD(65) (GAD-alum) treatment of children and adolescents with type 1 diabetes after 4 years of follow-up. METHODS: Seventy children and adolescents aged 10-18 years with recent onset type 1 diabetes participated in a phase II, double-blind, randomised placebo-controlled clinical trial. Patients identified as possible participants attended one of eight clinics in Sweden to receive information about the study and for an eligibility check, including a medical history. Participants were randomised to one of the two treatment groups and received either a subcutaneous injection of 20 µg of GAD-alum or placebo at baseline and 1 month later. The study was blinded to participants and investigators until month 30. The study was unblinded at 15 months to the sponsor and statistician in order to evaluate the data. At follow-up after 30 months there was a significant preservation of residual insulin secretion, as measured by C-peptide, in the group receiving GAD-alum compared with placebo. This was particularly evident in patients with <6 months disease duration at baseline. There were no treatment-related serious adverse events. We have now followed these patients for 4 years. Overall, 59 patients, 29 who had been treated with GAD-alum and 30 who had received placebo, gave their informed consent. RESULTS: One patient in each treatment group experienced an episode of keto-acidosis between months 30 and 48. There were no treatment-related adverse events. The primary efficacy endpoint was the change in fasting C-peptide concentration from baseline to 15 months after the prime injection for all participants per protocol set. In the GAD-alum group fasting C-peptide was 0.332 ± 0.032 nmol/l at day 1 and 0.215 ± 0.031 nmol/l at month 15. The corresponding figures for the placebo group were 0.354 ± 0.039 and 0.184 ± 0.033 nmol/l, respectively. The decline in fasting C-peptide levels between day 1 and month 1, was smaller in the GAD-alum group than the placebo group. The difference between the treatment groups was not statistically significant. In those patients who were treated within 6 months of diabetes diagnosis, fasting C-peptide had decreased significantly less in the GAD-alum group than in the placebo-treated group after 4 years. CONCLUSION/INTERPRETATION: Four years after treatment with GAD-alum, children and adolescents with recent-onset type 1 diabetes continue to show no adverse events and possibly to show clinically relevant preservation of C-peptide. TRIAL REGISTRATION: ClinicalTrials.gov NCT00435981 FUNDING: The study was funded by The Swedish Research Council K2008-55X-20652-01-3, Barndiabetesfonden (The Swedish Child Diabetes Foundation), the Research Council of Southeast Sweden, and an unrestricted grant from Diamyd Medical AB.

Microsomal prostaglandin E synthase-1 and 5-lipoxygenase: potential drug targets in cancer.

There is strong evidence for a role of prostaglandin (PG)E(2) in cancer cell proliferation and tumour development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by PGE synthases, including microsomal PGE synthase-1 (MPGES-1). Data describing genetic deletions of MPGES-1 are reviewed. The results suggest that MPGES-1 is an alternative therapeutic target for cancer cells and tumours that express this enzyme. Several compounds that target COX-2 or MPGES-1 also inhibit 5-lipoxygenase. This may be advantageous for treatment of some forms of cancer.

Expression of microsomal glutathione S-transferase type 3 mRNA in the rat nervous system.

Microsomal glutathione S-transferase type 3 (MGST3) is a recently identified member of a large superfamily of enzymes involved in biotransformation of xenobiotics and biosynthesis of eicosanoids, including prostaglandins and leukotrienes. Using in situ hybridization histochemistry and reverse transcription polymerase chain reaction, we characterized the expression of MGST3 mRNA in the rat nervous system based on the cloned rat MGST3 gene, under normal conditions and after systemic administration of lipopolysaccharide (LPS). The MGST3 mRNA seemed to be confined to neurons. The broad distribution in the brain was characterized by a strong signal in the hippocampal formation and in the nuclei of the cranial nerves. A moderate signal was found in the cortex, thalamus, amygdala and substantia nigra and a weak signal in the hypothalamus. Motoneurons in the spinal cord and sensory neurons in dorsal root ganglia displayed strong MGST3 mRNA signal. No significant changes in the level of expression of MGST3 mRNA in the brain were found 1, 3 or 6 h after LPS administration. The pattern of distribution of MGST3 mRNA in the rat nervous system and the lack of response to LPS do not support a role for MGST3 in the biosynthesis of proinflammatory eicosanoids but rather suggest other functions, perhaps in metabolic detoxication and neuroprotection.

Attenuation of the virulence of Porphyromonas gingivalis by using a specific synthetic Kgp protease inhibitor.

The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.

Resistance profiles of cyclic and linear inhibitors of HIV-1 protease.

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

Solid-phase synthesis of libraries generated from a 4-phenyl-2-carboxy-piperazine scaffold.

Strategies for finding novel structures of therapeutical interest are discussed. The rationale for the selection of the two scaffolds N4-(m-aminophenyl)-piperazine-2-carboxylic acid E and N4-(o-aminophenyl)-piperazine-2-carboxylic F is described. The synthesis of the appropriate precursors to scaffold E and F and their use in solid-phase chemistry are described. A 160-member library was produced combining these novel piperazine scaffolds with eight sulfonyl chlorides/acid chlorides and 10 amines. The compound library prepared was analyzed using LC-MS, showing the expected base peak in all wells at an average purity of 82%.

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Synthesis of potent C(2)-symmetric, diol-based hiv-1 protease inhibitors. Investigation of thioalkyl and thioaryl P1/P1' substituents.

The synthesis of novel, potent diol-based HIV-1 protease inhibitors, having either -SAr, -SCH(2)Ar, or -SCH(2)R groups as P1/P1' substituents is described. They can be prepared using a straightforward synthesis involving a thiol nucleophilic ring opening of a diepoxide. Inhibitor 13 was found to be a potent inhibitor of HIV-1 PR, showing good antiviral activity in a cell-based assay.

Synthesis of novel, potent, diol-based HIV-1 protease inhibitors via intermolecular pinacol homocoupling of (2S)-2-benzyloxymethyl-4-phenylbutanal.

The synthesis of novel, potent, diol-based HIV-1 protease inhibitors, having phenethyl groups (-CH(2)CH(2)Ph) in P1/P1' position is described. An intermolecular pinacol homocoupling of (2S)-2-benzyloxymethyl-4-phenylbutanal 16 was the key step in the synthesis. From this reaction sequence four carba analogues, compounds 8a, 8b, 9a, and 9b, were prepared, having the inverted configuration of one or both of the stereogenic centers carrying the diol hydroxyls as compared to the parent series represented by inhibitors 6 and 7. Inhibitor 8b was found to be a potent inhibitor of HIV-1 protease (PR), showing excellent antiviral activity in the cell-based assay and in the presence of 40% human serum. The absolute stereochemistry of the central diol of the potent inhibitor (8b) was determined from the X-ray crystallographic structure of its complex with HIV-1 PR.

Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.

Design and synthesis of potent C(2)-symmetric diol-based HIV-1 protease inhibitors: effects of fluoro substitution.

Implementation of derivatized carbohydrates as C(2)-symmetric HIV-1 protease inhibitors has previously been reported. With the objective of improving the anti-HIV activity of such compounds, we synthesized a series of fluoro substituted P1/P1' analogues. These compounds were evaluated for antiviral activity toward both wild type and mutant virus. The potency of the analogues in blocking HIV-1 protease was moderate, with K(i) values ranging from 1 to 7 nM. Nonetheless, compared to the parent nonfluorous inhibitors, a majority of the compounds exhibited improved antiviral activity, for example the 3-fluorobenzyl derivative 9b, which had a K(i) value of 7.13 nM and displayed one of the most powerful antiviral activities in the cellular assay of the series. Our results strongly suggest that fluoro substitution can substantially improve antiviral activity. The X-ray crystal structures of two of the fluoro substituted inhibitors (9a and 9f) cocrystallized with HIV-1 protease are discussed.

Cyclooxygenase-1 and cyclooxygenase-2 expression in rat kidney and adrenal gland after stimulation with systemic lipopolysaccharide: in situ hybridization and immunocytochemical studies.

Cyclooxygenase-2 (COX-2) is a recently discovered isoform of cyclooxygenase that is inducible by various types of inflammatory stimuli. Although this enzyme is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins, the precise location, distribution, and regulation of prostaglandin synthesis remains unclear in several tissues. Using in situ hybridization histochemistry, we investigated the induction of COX-1 and COX-2 mRNA expression after systemic administration of a pyrogen, lipopolysaccharide (LPS), in kidney and adrenal gland in the rat. The COX-2 mRNA signals dramatically increased 1 h after LPS treatment in the kidney outer medulla and adrenal cortex, where almost no or little expression was observed in nontreated animals, and returned to control levels within 24 h. COX-2 mRNA levels increased in the kidney inner medulla 6 h after treatment. There was also a significant increase in mRNA levels in the kidney cortex and adrenal medulla. On the other hand, COX-1 mRNA levels did not show any detectable changes except in the kidney inner medulla, where a significant downregulation of mRNA expression was observed after LPS treatment. Light and electron immunocytochemistry using COX-2 antibodies showed that strong COX-2 immunoreactivity was localized to certain cortical cells of the thick ascending limb of Henle. In addition, based on double-staining with antiserum to nitric oxide synthase (NOS) four further cell populations could be identified in kidney cortex, including weakly COX-2-positive, NOS-positive macula densa cells. After LPS treatment, changes in COX-2 immunoreactivity could be observed in interstitial cells in the kidney medulla and in inner cortical cells in the adrenal gland. These results show that COX-2 is a highly induced enzyme that can be up-regulated in specific cell populations in kidney and adrenal gland in response to inflammation, leading to the elevated levels of prostaglandins seen during fever. In contrast COX-1 mRNA levels remained unchanged in this experimental situation, except for a decrease in kidney inner medulla.

Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes.

The leukotrienes are inflammatory mediators derived from arachidonic acid. It was demonstrated that the priming of leukocytes with phorbol-12-myristate-13-acetate (PMA) leads to the increased formation of 5-lipoxygenase (5-LO) products in parallel with the increased association of 5-LO with the nucleus and the activation of kinases that can phosphorylate 5-LO in vitro. Stimulation of the monocytic cell line Mono Mac 6 with calcium ionophore gave low 5-LO product formation and no detectable redistribution of 5-LO. However, after priming of Mono Mac 6 cells with phorbol esters, ionophore led to the association of 45% to 75% of cellular 5-LO with the nuclear membrane, to 5-LO kinase activation, to enhanced release of arachidonate, and to substantial leukotriene synthesis. Similar results were obtained for human polymorphonuclear leukocytes stimulated with low-dose ionophore. In addition, for each cell type, PMA priming up-regulated leukotriene biosynthesis in the presence of exogenous arachidonic acid. A protein kinase inhibitor, calphostin C, reduced the association of 5-LO with the nucleus and 5-LO kinase activity, and the formation of 5-LO products was inhibited. These results suggest that PMA up-regulates leukotriene biosynthesis not only by increasing the release of endogenous arachidonate, but also by increasing the capacity for 5-LO phosphorylation and for the translocation of 5-LO to the nucleus in leukocytes.

IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression.

Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.

5-Lipoxygenase interacts with coactosin-like protein.

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics.

P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays.

The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1' benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 microM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1-100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.

Synthesis and comparative molecular field analysis (CoMFA) of symmetric and nonsymmetric cyclic sulfamide HIV-1 protease inhibitors.

We have previously reported on the unexpected flipped conformation in the cyclic sulfamide class of inhibitors. An attempt to induce a symmetric binding conformation by introducing P2/P2' substituents foreseen to bind preferentially in the S2/S2' subsite was unsuccessful. On the basis of the flipped conformation we anticipated that nonsymmetric sulfamide inhibitors, with P2/P2' side chains modified individually for the S1' and S2 subsites, should be more potent than the corresponding symmetric analogues. To test this hypothesis, a set of 18 cyclic sulfamide inhibitors (11 nonsymmetric and 7 symmetric) with different P2/P2' substituents was prepared and evaluated in an enzyme assay. To rationalize the structure-activity relationship (SAR) and enable the alignment of the nonsymmetric inhibitors, i.e., which of the P2/P2' substituents of the nonsymmetric inhibitors interact with which subsite, a CoMFA study was performed. The CoMFA model, constructed from the 18 inhibitors in this study along with seven inhibitors from previous work by our group, has successfully been used to rationalize the SAR of the cyclic sulfamide inhibitors. Furthermore, from the information presented herein, the SAR of the cyclic sulfamide class of inhibitors seems to differ from the SAR of the related cyclic urea inhibitors reported by DuPont and DuPont-Merck.

Characterization of a set of HIV-1 protease inhibitors using binding kinetics data from a biosensor-based screen.

The interaction between 290 structurally diverse human immunodeficiency virus type 1 (HIV-1) protease inhibitors and the immobilized enzyme was analyzed with an optical biosensor. Although only a single concentration of inhibitor was used, information about the kinetics of the interaction could be obtained by extracting binding signals at discrete time points. The statistical correlation between the biosensor binding data, inhibition of enzyme activity (K(i)), and viral replication (EC(50)) revealed that the association and dissociation rates for the interaction could be resolved and that they were characteristic for the compounds. The most potent inhibitors, with respect to K(i) and EC(50) values, including the clinically used drugs, all exhibited fast association and slow dissociation rates. Selective or partially selective binders for HIV-1 protease could be distinguished from compounds that showed a general protein-binding tendency by using three reference target proteins. This biosensor-based direct binding assay revealed a capacity to efficiently provide high-resolution information on the interaction kinetics and specificity of the interaction of a set of compounds with several targets simultaneously.

5-lipoxygenase expression in dendritic cells generated from CD34(+) hematopoietic progenitors and in lymphoid organs.

The 5-lipoxygenase (5-LO) pathway in human CD34(+) hematopoietic progenitor cells, which were induced to differentiate into dendritic cells (DCs) by cytokines in vitro and in DCs of lymphoid tissues in situ, was examined. Extracts prepared from HPCs contained low levels of 5-LO or 5-LO-activating protein. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor-alpha (TNF-alpha) promoted DC differentiation and induced a strong rise in 5-LO and FLAP expression. Fluorescence-activated cell sorter (FACS) analyses identified a major DC population coexpressing human leukocyte antigen (HLA)-DR/CD80 and monocytic or Langerhans cell markers. Transforming growth factor-beta1 (TGF-beta-1), added to support DC maturation, strongly promoted the appearance of CD1a(+)/Lag(+) Langerhans-type cells as well as mature CD83(+) DCs. TGF-beta-1 further increased 5-LO and FLAP expression, recruited additional cells into the 5-LO(+) DC population, and promoted production of 5-hydroxyeicosatetraenoic acid and leukotriene B(4) in response to calcium (Ca(++)) ionophore A23187. These in vitro findings were corroborated by 5-LO expression in distinct DC phenotypes in vivo. Scattered 5-LO and FLAP in situ hybridization signals were recorded in cells of paracortical T-lymphocyte-rich areas and germinal centers (GCs) of lymph nodes (LNs) and tonsil and in cells of mucosae overlying the Waldeyer tonsillar ring. 5-LO protein localized to both CD1a(+) immature DCs and to CD83(+) mature interdigitating DCs of T-lymphocyte-rich areas of LNs and tonsil. As DCs have the unique ability to initiate naive lymphocyte activation, our data support the hypothesis that leukotrienes act at proximal steps of adaptive immune responses. (Blood. 2000;96:3857-3865)

Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.