Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts... more
Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of ...
Research Interests:
Research Interests:
ABSTRACT
Research Interests:
We studied the effects of leukotrienes on in vitro functions of neutrophil polymorphonuclear (PMN) granulocytes. Leukotriene B4 (LTB4) evoked a stimulated and directed migration of neutrophils under agarose with an optimum concentration... more
We studied the effects of leukotrienes on in vitro functions of neutrophil polymorphonuclear (PMN) granulocytes. Leukotriene B4 (LTB4) evoked a stimulated and directed migration of neutrophils under agarose with an optimum concentration of 10(-6)M, whereas two nonenzymatically formed isomers (compounds I and II) induced this response at 10(-5)M. Leukotriene C4 (LTC4) and 5-hydroxyeicosate-traenoic acid (5-HETE) did not affect this PMN migration. At the same optimum concentrations, LTB4 and compounds I and II augmented PMN adherence to nylon fibers. The chemotactic and adherence responses were of the same magnitude as with formal-Met-Leu-Phe (fMLP) at 10(-7)M. None of the leukotrienes influenced the spontaneous or phagocytosis-associated chemiluminescence or the ability to kill Staphylococcus aures. The cyclooxygenase inhibitor, indomethacin, inhibited only partly the fMLP-induced migration at high concentrations and stimulated migration at 2.5 x 10(-7)M, suggesting that arachidonic ...
Research Interests:
Leukotrienes B4, C4, D4 and E4, together with five monohydroxyeicosatetraenoic acids, were isolated after incubation of chopped rat brain tissue with ionophore A23187. The monohydroxyeicosatetraenoic acids were... more
Leukotrienes B4, C4, D4 and E4, together with five monohydroxyeicosatetraenoic acids, were isolated after incubation of chopped rat brain tissue with ionophore A23187. The monohydroxyeicosatetraenoic acids were 5-hydroxy-6,8,11,14-eicosatetraenoic acid, 9-hydroxy-5,7,11,14-eicosatetraenoic acid, 11-hydroxy-5,8,12,14-eicosatetraenoic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Identification of the compounds was performed using reversed-phase high-performance liquid chromatography, ultraviolet spectroscopy and gas chromatography-mass spectrometry. Formation of the compounds was inhibited by micromolar concentrations of nordihydroguaiaretic acid. Indomethacin specifically inhibited the formation of 11-hydroxy-5,8,12,14-eicosatetraenoic acid, suggesting that this compound was produced as a by-product during cyclooxygenase-catalyzed prostaglandin synthesis.
Research Interests:
Leukotriene (LT) A4 hydrolase/aminopeptidase is a bifunctional zinc enzyme that catalyzes the final step in the biosynthesis of LTB4, a potent chemoattractant and immune modulating lipid mediator. Here, we report a high-resolution crystal... more
Leukotriene (LT) A4 hydrolase/aminopeptidase is a bifunctional zinc enzyme that catalyzes the final step in the biosynthesis of LTB4, a potent chemoattractant and immune modulating lipid mediator. Here, we report a high-resolution crystal structure of LTA4 hydrolase in complex with captopril, a classical inhibitor of the zinc peptidase angiotensin-converting enzyme. Captopril makes few interactions with the protein, but its free thiol group is bound to the zinc, apparently accounting for most of its inhibitory action on LTA4 hydrolase. In addition, we have determined the structures of LTA4 hydrolase in complex with two selective tight-binding inhibitors, a thioamine and a hydroxamic acid. Their common benzyloxyphenyl tail, designed to mimic the carbon backbone of LTA4, binds into a narrow hydrophobic cavity in the protein. The free hydroxyl group of the hydroxamic acid makes a suboptimal, monodentate complex with the zinc, and strategies for improved inhibitor design can be deduced from the structure. Taken together, the three crystal structures provide the molecular basis for the divergent pharmacological profiles of LTA4 hydrolase inhibitors. Moreover, they help define the binding pocket for the fatty acid-derived epoxide LTA4 as well as the subsites for a tripeptide substrate, which in turn have important implications for the molecular mechanisms of enzyme catalyses.
Research Interests: Physiology, Catalysis, Enzyme Inhibitors, Macromolecular X-Ray Crystallography, Molecular Mechanics, and 16 moreCrystal structure, Amines, Zinc, Immune modulation, Medical Physiology, Enzyme, High Resolution, Tight Binding, Fatty Acid, Protein Conformation, Angiotensin Converting Enzyme, Protein Binding, Hydroxamic Acids, Biochemistry and cell biology, Molecular Structure, and Captopril
RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1+)... more
RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1+) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximately 23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1+ was required for silencing of two genes containing a conserved motif.
Research Interests:
5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we... more
5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinu...
Research Interests:
5-Lipoxygenase (5-LOX) catalyzes two steps in the biosynthesis of leukotrienes (LTs), lipid mediators of inflammation derived from arachidonic acid. In this review we focus on 5-LOX biochemistry including 5-LOX interacting proteins and... more
5-Lipoxygenase (5-LOX) catalyzes two steps in the biosynthesis of leukotrienes (LTs), lipid mediators of inflammation derived from arachidonic acid. In this review we focus on 5-LOX biochemistry including 5-LOX interacting proteins and regulation of enzyme activity. LTs function in normal host defense, and have roles in many disease states where acute or chronic inflammation is part of the pathophysiology, as briefly summarized at the end of this chapter. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".
Research Interests:
A cDNA encoding an arachidonate 12-lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12-lipoxygenase cDNA were used to isolate the initial polymerase-chain-reaction... more
A cDNA encoding an arachidonate 12-lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12-lipoxygenase cDNA were used to isolate the initial polymerase-chain-reaction product (395 bp). The final sequence of the rat 12-lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase-chain-reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75,305 Da. Also the rat 12-lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12-lipoxygenase (71%) and to human 15-lipoxygenase (75%), with less resemblance to human platelet 12-lipoxygenase (59%) or rat leukocyte 5-lipoxygenase (41%). The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12-lipoxygenase (but also some 15-lipoxygenase) enzyme activity was obtained. A part of the brain 12-lipoxygenase cDNA was used as probe in Northern blots. A 2.7-kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12-lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.
Research Interests:
Research Interests:
Research Interests:
Arachidonic acid is released from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores) and converted to leukotrienes by a 5-lipoxygenase that also has leukotriene A4 synthetase activity.... more
Arachidonic acid is released from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores) and converted to leukotrienes by a 5-lipoxygenase that also has leukotriene A4 synthetase activity. Leukotriene A4, an unstable epoxide, is hydrolyzed to leukotriene B4 or conjugated with glutathione to yield leukotriene C4 and its metabolites, leukotriene D4 and leukotriene E4. The leukotrienes participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. Recent studies also suggest a neuroendocrine role for leukotriene C4 in luteinizing hormone secretion. Lipoxins are formed by the action of 5- and 15-lipoxygenases on arachidonic acid. Lipoxin A causes contraction of guinea pig lung strips and dilation of the microvasculature. Both lipoxin A and B inhibit natural killer cell cytotoxicity. Thus, the multiple interaction of lipoxygenases generates compounds that can regulate specific cellular responses of importance in inflammation and immunity.
Research Interests:
ABSTRACT
Research Interests:
Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in... more
Lesional epidermis of psoriasis has a probable reduction in the cyclic AMP/cyclic GMP ratio. This altered ratio may in part be responsible for the characteristic glycogen storage, rapid cell proliferation, and reduced differentiation in lesional epidermis. The concentrations of prostaglandins E2 and F2alpha, free arachidonic acid, and 12L-hydroxy-5,8,10,14-eicosatetrawnoic acid in specimens of uninvolved and involved epidermis of psoriasis were measured with deuterium-labeled carriers and multiple ion analysis. Snap frozen specimens contained: 1.4 +/- 0.4 mug/g (wet weight) of arachidonic acid in uninvolved in contrast to 36.3 +/- 16.7 mug/g in involved epidermis (P = 0.015); less than 0.05 +/- 0.01 mug/g of hydroxyeicosatetraenoic acid in uninvolved in contrast to 4.1 +/- 1.9 mug/g in involved epidermis (P = 0.015); 23.6 +/- 5.0 ng/g of prostaglandin E2 in uninvolved in contrast to 33.1 +/- 5.7 ng/g in involved epidermis (P less than 0.01); and 21.0 +/- 4.4 ng/g of prostaglandin F2alpha in uninvolved in contrast to 39.0 +/- 5.9 ng/g in involved epidermis (P less than 0.01). The arachidonic acid and hydroxyeicosatetraenoic acid levels in involved epidermis were strongly correlated (r = 0.97). The increased levels of arachidonic acid and 12L-hydroxy-5,8,10,14-eicosatetraenoic acid in involved epidermis may have diagnostic and pathophysiological importance.
Research Interests:
Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous... more
Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was expressed in Escherichia coli, and both cytosolic and membrane fractions were prepared. Western blot analysis specifically detected a 15- to 16-kDa protein in the membrane fraction. Both fractions were incubated with prostaglandin H2 in the presence or absence of reduced glutathione. The membrane but not the cytosolic fraction was found to possess high glutathione-dependent PGE synthase activity (0.25 micromol/min/mg). The human tissue distribution was analyzed by Northern blot analysis. High expression of PGE synthase mRNA was detected in A549 and HeLa cancer cell lines. Intermediate level of expression was demonstrated in placenta, prostate, testis, mammary gland, and bladder whereas low mRNA expression was observed in several other tissues. A549 cells have been used as a model system to study cyclooxygenase-2 induction by IL-1beta. If A549 cells were grown in the presence of IL-1beta, a significant induction of the PGE synthase was observed by Western blot analysis. Also, Western blot analysis specifically detected a 16-kDa protein in sheep seminal vesicles. In summary, we have identified a human membrane bound PGE synthase. The enzyme activity is glutathione-dependent, and the protein expression is induced by the proinflammatory cytokine IL-1beta. PGE synthase is a potential novel target for drug development.
Research Interests: Drug development, Multidisciplinary, Cell line, Humans, Escherichia coli, and 17 moreGlutathione, Drug Design, Drug Delivery Systems, Enzyme, mRna expression levels, HeLa cells, Mammary gland, Model System, Western blot, Protein Expression, Enzyme activity, Amino Acid Sequence, Base Sequence, Drug Targeting, Prostaglandin E2, Interleukin, and Enzyme Induction
Research Interests:
When arachidonic acid was incubated with homogenates of potato tubers, two isomers of 6-trans-leukotriene B4, epimeric at C-12, were formed in addition to the major product, (5S-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid... more
When arachidonic acid was incubated with homogenates of potato tubers, two isomers of 6-trans-leukotriene B4, epimeric at C-12, were formed in addition to the major product, (5S-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE). To elucidate the mechanism of biosynthesis of the dihydroxy-acids, the lipoxygenase from the potato tubers was purified to apparent homogeneity by a combination of conventional chromatographic procedures and high-performance liquid chromatography equipped with a chromatofocusing column (Mono-P). The purified lipoxygenase acted on arachidonic acid and bishomo-gamma-linolenic acid to yield (5S)-hydroperoxy- and (8S)-hydroperoxyicosanoids, respectively. Furthermore, the purified enzyme converted 5-HPETE to leukotriene A4, with the presence of the epoxide intermediate being demonstrated by 18O2 experiments, methanol trapping, as well as further conversion to leukotriene B4 by the purified leukotriene A4 hydrolase. Several experiments, including those with lipoxygenase inhibitors, heat treatment, and competitive inhibition, indicated that both the 5-lipoxygenase and leukotriene A4 synthase activities resided in the same protein and that the formation of leukotriene A4 from 5-HPETE was catalyzed by the 8-lipoxygenase activity of the enzyme.
Research Interests:
Leukotrienes B4, C4, and D4, members of a recently discovered family of substances biosynthesized from arachidonic acid, were found to have potent microvascular actions in the hamster cheek pouch. When applied topically to the vascular... more
Leukotrienes B4, C4, and D4, members of a recently discovered family of substances biosynthesized from arachidonic acid, were found to have potent microvascular actions in the hamster cheek pouch. When applied topically to the vascular network, leukotrienes C4 and D4 caused an intense constriction of arterioles, being similar to angiotensin in potency in this respect. The vasoconstriction induced by leukotrienes C4 and D4 was short-lived, and it was consistently followed by a marked and dose-dependent extravasation of macromolecules from postcapillary venules. Histamine did not constrict arterioles, but it elicited leakage of plasma, although on a molar basis it was no more than 1/1000th as potent as the leukotrienes. When used in the same concentration range as leukotrienes C4 and D4, leukotriene B4 did not evoke vasoconstriction or promote plasma leakage. On the other hand, leukotriene B4 caused a conspicuous and reversible adhesion of leukocytes to the endothelium in postcapillary venules. Our findings that leukotrienes induce microcirculatory alterations in vivo, closely resembling the early events in the acute inflammatory response, imply that leukotrienes, formed in several blood-borne and tissue-bound cells, may mediate important microcirculatory adjustments to noxious stimuli.
Research Interests:
Research Interests:
A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial... more
A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.
Research Interests: Kinetics, Multidisciplinary, DNA, Protein Structure and Function, Cell line, and 21 moreCell Differentiation, Humans, Escherichia coli, DNA hybridization, DNA sequence design, Enzyme, Molecular cloning, Amino Acid Profile, Molecular weight, Codon, Amino Acid Sequence, Base Sequence, Open Reading Frame, Organic Compound, Phorbol ester, Blood cells, Full Length Movies, cDNA library, Molecular Structure, Nucleic Acid, and Enzyme Induction
Methods were developed for quantitative determination of the three major metabolites of arachidonic acid in human platelets, i.e., 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and... more
Methods were developed for quantitative determination of the three major metabolites of arachidonic acid in human platelets, i.e., 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (PHD). Aggregation of washed platelets by thrombin was accompanied by release of 1163-2175 ng/ml of HETE, 1129-2430 ng/ml of HHT, and 998-2299 ng/ml of PHD. The amount of PGG(2) (prostaglandin G(2)) produced as calculated from the sum of the amounts of its metabolites (HHT and PHD) was 2477-5480 ng/ml. In contrast, the amounts of PGE(2) (prostaglandin E(2)) and PGF(2alpha) (prostaglandin F(2alpha)) released were approximately two orders of magnitude lower. In this system, the prostaglandins thus exert their biological action through the endoperoxides, which are almost exclusively metabolized to nonprostanoate structures and only to a small extent to the classical prostaglandins. Platelets from subjects given aspirin produced less than 5% of the above mentioned amounts of HHT and PHD, whereas the production of HETE was stimulated about 3-fold. This provides additional evidence for our earlier proposal [Hamberg, M., Svensson, J., Wakabayashi, T. & Samuelsson, B. (1974) Proc. Nat. Acad. Sci. USA 71, 345-349] that the anti-aggregating effect of aspirin is through inhibition of PGG(2) formation.
Research Interests:
Research Interests: Caenorhabditis elegans, Multidisciplinary, Saccharomyces cerevisiae, Cell line, Humans, and 14 moreSequence alignment, Animals, Dictyostelium, Lung, Molecular cloning, Protein Interaction, Hybrid Approach, Transforming Growth Factor Beta, Protozoan Proteins, Amino Acid Sequence, Base Sequence, Recombinant Proteins, double-stranded RNA, and cDNA library
Research Interests: Leukemia, Multidisciplinary, Animals, Sulfur, Amino Acids, and 3 moreRats, Guinea Pig, and Amino Acid Profile
Research Interests:
Research Interests:
We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer,... more
We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.
Research Interests:
Research Interests:
5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human... more
5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.
Research Interests:
Research Interests:
Abstract Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was... more
Abstract Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two ...
Research Interests:
Research Interests:
Research Interests:
Incubation for a short time of arachidonic acid with the microsomal fraction of a homogenate of the vesicular gland of sheep in the presence of 1 mM p-mercuribenzoate followed by extraction and silicic acid chromatography yielded two... more
Incubation for a short time of arachidonic acid with the microsomal fraction of a homogenate of the vesicular gland of sheep in the presence of 1 mM p-mercuribenzoate followed by extraction and silicic acid chromatography yielded two prostaglandin endoperoxides. The structures of these compounds, i.e., 15-hydroperoxy-9alpha,11alpha-peroxidoprosta-5,13-dienoic acid (prostaglandin G(2)) and 15-hydroxy-9alpha,11alpha-peroxidoprosta-5,13-dienoic acid (prostaglandin H(2)), were assigned mainly by a number of chemical transformations into previously known prostaglandins. The new prostaglandins were 50-200 times (prostaglandin G(2)) and 100-450 times (prostaglandin H(2)) more active than prostaglandin E(2) on the superfused aorta strip. The half-life of the prostaglandin endoperoxides in aqueous medium (about 5 min) was significantly longer than that of "rabbit aorta-contracting substance" released from guinea pig lung, indicating that none of the prostaglandin endoperoxides is identical with this factor. Addition of 10-300 ng/ml of the endoperoxides to suspensions of washed human platelets resulted in rapid aggregation. Furthermore, platelet aggregation induced by thrombin was accompanied by release of material reducible by stannous chloride into prostaglandin F(2alpha), thus indicating the involvement of endogenous prostaglandin endoperoxides in platelet aggregation.
Research Interests:
The endoperoxide prostaglandin G2 (PGG2) induced platelet aggregation as well as the platelet release reaction (release of ADP and serotonin) when added to human platelet-rich plasma. Formation of a metabolite of PGG2... more
The endoperoxide prostaglandin G2 (PGG2) induced platelet aggregation as well as the platelet release reaction (release of ADP and serotonin) when added to human platelet-rich plasma. Formation of a metabolite of PGG2 [8-(l-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid] and a lipoxygenase product [12L-hydroxy-5,8,10,14-eicosatetraenoic acid] accompanied the release reaction caused by aggregating agents such as collagen, ADP, epinephrine, and thrombin. Indomethacin inhibited the release reaction and PGG2 formation induced by these agents but had no effect on PGG2-induced release reaction. The aggregating effect of PGG2 was abolished by furosemide, which is a competitive inhibitor of ADP-induced primary aggregation. These data indicate that the aggregating effect of PGG2 is due to release of ADP and that PGG2 synthesis is required for induction of the release reaction by various aggregating agents. A subject with a hemostatic defect due to abnormal release mechanism [decreased aggregation with epinephrine (second wave) and collagen and normal platelet ADP] had a deficiency of the cyclo-oxygenase that catalyzes formation of PGG2. Normal aggregation and release reaction were obtained with added PGG2. Ii is concluded that the endoperoxide (PGG2) is essential in normal hemostasis because of its role in initiating the release reaction required for aggregation by collagen and the second wave of aggregation caused by, e.g., ADP.
Research Interests:
Arachidonic acid incubated with human platelets was converted into three compounds, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, 12L-hydroxy-5,8,10-heptadecatrienoic acid, and the hemiacetal derivative of... more
Arachidonic acid incubated with human platelets was converted into three compounds, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, 12L-hydroxy-5,8,10-heptadecatrienoic acid, and the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid. The formation of the two latter compounds from arachidonic acid proceeded by pathways involving the enzyme, fatty acid cyclo-oxygenase, in the initial step and with the prostaglandin endoperoxide, PGG(2), as an intermediate. The first mentioned compound was formed from 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which in turn was formed from arachidonic acid by the action of a novel lipoxygenase. Aspirin and indomethacin inhibited the fatty acid cyclo-oxygenase but not the lipoxygenase, whereas 5,8,11,14-eicosatetraynoic acid inhibited both enzymes. The almost exclusive transformation of the endoperoxide structure into non-prostaglandin derivatives supports the hypothesis that the endoperoxides can participate directly and not by way of the classical prostaglandins in regulation of cell functions. The observed transformations of arachidonic acid in platelets also explain the aggregating effect of this acid.
Research Interests:
Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli. In incubations of E. coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and... more
Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli. In incubations of E. coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene A4 were formed, while incubation with 8,11,14-eicosatrienoic acid gave 8-hydroxy-9,11,14-eicosatrienoic acid. Six conserved histidine residues in 5-lipoxygenase were subjected to site-directed mutagenesis. Exchanges of His-367, -372, or -551 gave mutants for which no enzyme activities were detectable. On the other hand, exchanges of His-362, -390, or -399 gave mutants that were enzymatically active, but less so than the nonmutated control. For two of these (exchanges of His-390 or -399), the activities of the mutants were dependent on the expression temperature. Thus, the histidines in the first group (His-367, -372, -551) were crucial for 5-lipoxygenase activity, possibly because of a function of these residues as metal ligands. Mutagenesis aimed at two other conserved elements in 5-lipoxygenase, Gln-558 and the C terminus, gave mutated proteins with only a small residual activity (substitution of Gln-558), or with no detectable activity (deletion of six C-terminal amino acids), indicating that these regions are important for the function of 5-lipoxygenase.
Research Interests: Kinetics, Gene expression, Multidisciplinary, Western blotting, Humans, and 11 moreEscherichia coli, Substrate Specificity, Enzyme activity, Amino Acid Profile, Base Sequence, Structure activity Relationship, Recombinant Proteins, Enzymatic Activity, Site-directed Mutagenesis, Histidine, and Arachidonic Acid
Abstract 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,... more
Abstract 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 α. The ...
Research Interests:
A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11... more
A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes.
Research Interests:
An earlier proposed endoperoxide intermediate in the biosynthesis of prostaglandins was detected in short-time incubations of arachidonic acid with the microsomal fraction of homogenates of sheep vesicular glands. Conversion of the... more
An earlier proposed endoperoxide intermediate in the biosynthesis of prostaglandins was detected in short-time incubations of arachidonic acid with the microsomal fraction of homogenates of sheep vesicular glands. Conversion of the endoperoxide into prostaglandin E(2) was stimulated by reduced glutathione but suppressed by p-mercuribenzoate and N-ethylmaleimide. The methyl ester of an unknown compound was isolated by solvent extraction and thin-layer chromatography after short-time incubation of arachidonic acid with the microsomal fraction and p-mercuribenzoate. This derivative was identical to the methylester of the endoperoxide, as shown by its conversion into the methyl esters of 11-dehydroprostaglandin F(2alpha) and prostaglandin E(2) by spontaneous rearrangement and its conversion into the methyl ester of prostaglandin F(2alpha) by mild chemical reduction. The smooth muscle-stimulating activity of the endoperoxide ester on the isolated rabbit aortas trip was 4- to 8-times higher than that of the methyl ester of prostaglandin E(2).
Research Interests:
Research Interests: Multidisciplinary, Humans, Animals, DNA sequence design, Chemical Analysis, and 15 morePlasmids, Transcription Factor, Enzyme, Genes, Introns, Molecular cloning, HeLa cells, Transfection, Rabbits, RNA Structure, Amino Acid Sequence, Base Sequence, Sperm Dna Fragmentation, Organic Compound, and Nucleic Acid
An unstable [t1/2 at 37 degrees = 32 +/- 2 (SD) sec] intermediate, thromboxane A2, was detected in the conversion of prostaglandin G2 into 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2) in platelets.... more
An unstable [t1/2 at 37 degrees = 32 +/- 2 (SD) sec] intermediate, thromboxane A2, was detected in the conversion of prostaglandin G2 into 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2) in platelets. The intermediate was trapped by addition of methanol, ethanol, or sodium azide to suspensions of washed human platelets incubated for 30 sec with arachidonic acid or prostaglandin G2. The structures of the resulting derivatives demonstrated that the intermediate possessed an oxane ring as in thromboxane B2 but lacked its hemiacetal hydroxyl group. Additional experiments using 18O2 or [2H8]arachidonic acid in the formation of thromboxane B2 and CH3O2H for the trapping of thromboxane A2, together with information on the t1/2 of the intermediate, indicated the presence of an oxetane structure in thromboxane A2. Incubation of arachidonic acid or prostaglandin G2 with washed platelets led to formation of an unstable factor that induced irreversible platelet aggregation and caused release of [14C]serotonin from platelets that had been incubated with [14C]serotonin. The properties and the mode of formation of this factor indicated that it was identical with thromboxane A2. Furthermore, evidence is presented that the more unstable and major component of rabbit aorta contracting substance (RCS) formed in platelets and guinea pig lung is also thromboxane A2.
Research Interests:
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.... more
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.