The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform
Soo Wan Nam - One of the best experts on this subject based on the ideXlab platform.
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Rapid purification of recombinant human Lipocortin-I secreted from Saccharomyces cerevisiae
Springer Verlag (Germany), 2000Co-Authors: Bong Hyun Chung, Soo Wan NamAbstract:Human Lipocortin-I was expressed as a secretory product by Saccharomyces cerevisiae harboring an expression system consisting of GAL10 promoter, inulinase signal sequence and Lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human Lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The Lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified Lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.ope
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High-level secretory production of recombinant human Lipocortin-I by Saccharomyces cerevisiae
Process Biochemistry, 1999Co-Authors: Bong Hyun Chung, Dong Jin Seo, Soo Wan NamAbstract:Abstract Human Lipocortin-I was very efficiently produced as a secretory product by Saccharomyces cerevisiae harbouring an optimized expression casette containing the GAL10 promoter, inulinase signal sequence and the Lipocortin-I terminator. To overproduce Lipocortin-I, a fed-batch fermentation was performed. The feed medium contained only glucose as sole carbon source and was first fed for cell growth. A mixture of glucose and galactose was then fed for gene induction in parallel with cell growth. During the gene induction period, a significant amount of Lipocortin-I accumulated in the culture medium. However, about 45% of the secreted Lipocortin-I existed in a proteolytically-cleaved form, cleaved after the basic amino acid Lys 26 . At the end of the culture, the concentration of total extracellular Lipocortin-I (intact form+cleaved form) was about 2.1 g/l, accounting for more than 80% of the total extracellular protein.
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High-level secretory production of recombinant human Lipocortin-I by Saccharomyces cerevisiae
Process Biochemistry, 1999Co-Authors: Bong Hyun Chung, Dong Jin Seo, Soo Wan NamAbstract:Human Lipocortin-I was very efficiently produced as a secretory product by Saccharomyces cerevisiae harbouring an optimized expression casette containing the GAL10 promoter, inulinase signal sequence and the Lipocortin-I terminator. To overproduce Lipocortin-I, a fed-batch fermentation was performed. The feed medium contained only glucose as sole carbon source and was first fed for cell growth. A mixture of glucose and galactose was then fed for gene induction in parallel with cell growth. During the gene induction period, a significant amount of Lipocortin-I accumulated in the culture medium. However, about 45% of the secreted Lipocortin-I existed in a proteolytically-cleaved form, cleaved after the basic amino acid Lys26. At the end of the culture, the concentration of total extracellular Lipocortin-I (intact form + cleaved form) was about 2.1 g/l, accounting for more than 80% of the total extracellular protein.ope
Bong Hyun Chung - One of the best experts on this subject based on the ideXlab platform.
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Rapid purification of recombinant human Lipocortin-I secreted fromSaccharomyces cerevisiae
Biotechnology and Bioprocess Engineering, 2000Co-Authors: Bong Hyun ChungAbstract:Human Lipocortin-I was expressed as a secretory product by Saccharomyces cerevisiae harboring an expression system consisting of GAL10 promoter, inulinase signal sequence and Lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human Lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The Lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified Lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys^26.
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Rapid purification of recombinant human Lipocortin-I secreted from Saccharomyces cerevisiae
Springer Verlag (Germany), 2000Co-Authors: Bong Hyun Chung, Soo Wan NamAbstract:Human Lipocortin-I was expressed as a secretory product by Saccharomyces cerevisiae harboring an expression system consisting of GAL10 promoter, inulinase signal sequence and Lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human Lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The Lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified Lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.ope
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High-level secretory production of recombinant human Lipocortin-I by Saccharomyces cerevisiae
Process Biochemistry, 1999Co-Authors: Bong Hyun Chung, Dong Jin Seo, Soo Wan NamAbstract:Abstract Human Lipocortin-I was very efficiently produced as a secretory product by Saccharomyces cerevisiae harbouring an optimized expression casette containing the GAL10 promoter, inulinase signal sequence and the Lipocortin-I terminator. To overproduce Lipocortin-I, a fed-batch fermentation was performed. The feed medium contained only glucose as sole carbon source and was first fed for cell growth. A mixture of glucose and galactose was then fed for gene induction in parallel with cell growth. During the gene induction period, a significant amount of Lipocortin-I accumulated in the culture medium. However, about 45% of the secreted Lipocortin-I existed in a proteolytically-cleaved form, cleaved after the basic amino acid Lys 26 . At the end of the culture, the concentration of total extracellular Lipocortin-I (intact form+cleaved form) was about 2.1 g/l, accounting for more than 80% of the total extracellular protein.
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High-level secretory production of recombinant human Lipocortin-I by Saccharomyces cerevisiae
Process Biochemistry, 1999Co-Authors: Bong Hyun Chung, Dong Jin Seo, Soo Wan NamAbstract:Human Lipocortin-I was very efficiently produced as a secretory product by Saccharomyces cerevisiae harbouring an optimized expression casette containing the GAL10 promoter, inulinase signal sequence and the Lipocortin-I terminator. To overproduce Lipocortin-I, a fed-batch fermentation was performed. The feed medium contained only glucose as sole carbon source and was first fed for cell growth. A mixture of glucose and galactose was then fed for gene induction in parallel with cell growth. During the gene induction period, a significant amount of Lipocortin-I accumulated in the culture medium. However, about 45% of the secreted Lipocortin-I existed in a proteolytically-cleaved form, cleaved after the basic amino acid Lys26. At the end of the culture, the concentration of total extracellular Lipocortin-I (intact form + cleaved form) was about 2.1 g/l, accounting for more than 80% of the total extracellular protein.ope
Nancy J. Rothwell - One of the best experts on this subject based on the ideXlab platform.
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Lipocortin-1: cellular mechanisms and clinical relevance
Trends in pharmacological sciences, 1994Co-Authors: R.j. Flower, Nancy J. RothwellAbstract:Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of Lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant Lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of Lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of Lipocortin-1.
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Involvement of interleukin-1 and Lipocortin-1 in ischaemic brain damage.
Cerebrovascular and brain metabolism reviews, 1993Co-Authors: Nancy J. Rothwell, Jane K. ReltonAbstract:The cytokine interleukin-1 (IL-1) is synthesised within the brain and acts as a mediator of host defence responses to disease and injury. Several of these central actions of IL-1 are inhibited by an endogenous calcium and phospholipid binding protein, Lipocortin-1. Synthesis of IL-1 and Lipocortin-1 in the brain is markedly increased by neuronal damage, and inhibition of the actions of endogenous IL-1 by central injection of IL-1 receptor antagonist in the rat significantly inhibits ischaemic and excitotoxic brain damage. Lipocortin-1 appears to act as an endogenous neuroprotective agent that markedly attenuates ischaemic and excitotoxic damage. In contrast, inhibition of the actions of Lipocortin-1 by injection of neutralising antiserum exacerbates both forms of neurodegeneration. The mechanisms underlying these effects of IL-1 and Lipocortin-1 are largely unknown, but are probably independent of changes in body temperature. Actions of these molecules on corticotrophin releasing factor, arachidonic acid, excitatory amino acids, and nitric oxide, and the possible involvement of these factors in brain damage are discussed.
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Lipocortin-1 inhibits NMDA receptor-mediated neuronal damage in the striatum of the rat
Brain Research, 1992Co-Authors: Mark D. Black, Jane K. Relton, Frank Carey, Alan R. Crossman, Nancy J. RothwellAbstract:Lipocortin-1 (annexin-1), an endogenous phospholipid and calcium binding protein, has been shown to significantly attenuate the damage produced by focal cerebral ischaemia in the rat. In the present study we have therefore investigated its effect on N-methyl-D-aspartate (NMDA) induced neuronal damage. Unilateral intrastriatal infusion of a potent and selective NMDA agonist, cis-2,4-methanoglutamate (MGlu), induced an extensive lesion of the striatum in the rat, which was inhibited (greater than 80%) by prior injection of MK801 (4 mg/kg, i.p.). Infusion of 1.2 micrograms of an active fragment of Lipocortin-1 (N-terminal 1-188 aa) immediately after MGlu significantly reduced the extent of damage by 44.2 +/- 8.0%. In contrast, infusion of 3 microliters of neutralizing anti-Lipocortin-1 antibody with MGlu increased lesion size by 158.9 +/- 22.0%. These findings indicate that the damage produced by intrastriatal infusion of MGlu is mediated by the NMDA receptor. Lipocortin-1 fragment markedly attenuated, and the neutralizing antibody increased, this NMDA mediated neuronal damage. These observations may explain the neuroprotective action of Lipocortin following cerebral ischaemia.
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Lipocortin-1 is an endogenous inhibitor of ischemic damage in the rat brain.
The Journal of experimental medicine, 1991Co-Authors: Jane K. Relton, Paul J. L. M. Strijbos, Celestine T. O'shaughnessy, Frank Carey, Robert A. Forder, Fred J H Tilders, Nancy J. RothwellAbstract:Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated Lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of Lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of Lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The Lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of Lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antiLipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that Lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.
R.j. Flower - One of the best experts on this subject based on the ideXlab platform.
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antisense oligonucleotides to human Lipocortin 1 inhibit glucocorticoid induced inhibition of a549 cell growth and eicosanoid release
Biochemical Pharmacology, 1994Co-Authors: Jamie D Croxtall, R.j. FlowerAbstract:Glucocorticoids actively inhibit the growth of A549 cells by suppressing the release of factors such as prostaglandin E2 (PGE2) necessary for their proliferation. This effect is largely mediated through induction of the protein Lipocortin-1. We now show that transient transfection of A549 cells with an antisense DNA oligonucleotide targeted to a region coding the unique N-terminal portion of human Lipocortin-1 blocks the induction of Lipocortin-1 protein following glucocorticoid treatment and completely reverses glucocorticoid-induced suppression of cell proliferation and PGE2 release. A scrambled oligonucleotide was without effect. Continued culture of A549 cells in the presence of this oligonucleotide results in a sustained increase in cell proliferation and PGE2 release. This study reinforces the importance of Lipocortin-1 as a negative modulator of cell growth and eicosanoid generation in this system.
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Lipocortin-1: cellular mechanisms and clinical relevance
Trends in pharmacological sciences, 1994Co-Authors: R.j. Flower, Nancy J. RothwellAbstract:Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of Lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant Lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of Lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of Lipocortin-1.
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Lipocortins (annexins) 1, 2, 4 and 5 are increased in the central nervous system in multiple sclerosis.
Journal of neuroimmunology, 1992Co-Authors: A. J. Elderfield, J. Newcombe, C. Bolton, R.j. FlowerAbstract:Western blotting and densitometry have been used to investigate the Lipocortin content of post-mortem central nervous system (CNS) tissue samples from multiple sclerosis (MS) patients and normal controls. Lipocortins 1, 2, 4 and 5 were all detected in normal control grey and white matter. In white matter samples from MS patients these Lipocortins were found to be significantly increased, a further elevation in Lipocortin content was observed in MS plaque tissue. The implications of these findings with respect to the role of these proteins in inflammatory CNS disease and a possible mechanism of steroid action in the therapy of MS are discussed.
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Circulating autoantibodies to recombinant Lipocortin-1 in asthma
Respiratory medicine, 1991Co-Authors: Kian Fan Chung, N J Goulding, J.l. Godolphin, R.j. Flower, M.r. Podgorski, P.r. Sharland, B J O'connor, Peter J. BarnesAbstract:One of the postulated mechanisms of corticosteroid action is through the de novo synthesis and release of Lipocortins. We assayed circulating antibodies to Lipocortin-1 in sera obtained from normal ( n = 67) and asthmatic ( n = 57) subjects using an ELISA technique. Asthmatic subjects with a wide range of severity, with the mildest needing only occasional inhaled β-agonist therapy to the most severe needing maintenance oral corticosteroid treatment, were recruited from our Asthma Clinic and classified into five categories according to the need of therapy. Median values of IgM and IgG Lipocortin-1 antibody for normal subjects were 19·3 (interquartile range ( r ) = 11·0–30·4) and 16·9 ( r = 10·54–29·4) ELISA units (EU) ml −1 respectively. These levels were significantly elevated in asthmatic subjects: IgM=43·9 EU ml −1 ( r = 31·7–64·5) and IgG=29·0 EU ml −1 ( r = 21·2–44·7) ( P
Reiji Kannagi - One of the best experts on this subject based on the ideXlab platform.
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Immunohistochemical localization of Lipocortins in normal and psoriatic human skin
Archives of dermatological research, 1993Co-Authors: K. Ikai, Yoshihiro Ando, Sadao Imamura, Kyoko Shimizu, Fukumi Furukawa, Reiji KannagiAbstract:The distribution of Lipocortin I, a steroid-induced inhibitory protein of phospholipase A2, was examined in normal and psoriatic human skin. Using immunoblotting analysis with specific antibody against human Lipocortin I purified from human placenta, Lipocortin I was detected as a 37 kDa protein in cultured epidermal cells, whole skin and epidermis. In the dermis and stratum corneum, Lipocortin I was only weakly detectable by Western blotting. In contrast to normal skin, much less Lipocortin I was detected by Western blotting analysis in psoriatic skin. Using immunoperoxidase immunohistochemical analysis, Lipocortin I was demonstrated in the cytoplasm of keratinocytes in the upper and middle layers of the epidermis and in some infiltrating cells in the dermis in normal skin. In involved psoriatic skin, by contrast, Lipocortin I was almost undetectable in the epidermis, although it was demonstrated in some infiltrating cells in the dermis. No immunostaining of Lipocortin I was observed in the stratum corneum of normal or psoriatic skin. These results, together with the finding that phospholipase A2 activity is higher in psoriatic epidermis than in normal epidermis, suggest that Lipocortin I plays an important role in the regulation of differentiation and proliferation of epidermal keratinocytes.
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Calcium-induced intracellular cross-linking of Lipocortin I by tissue transglutaminase in A431 cells : augmentation by membrane phospholipids
The Journal of biological chemistry, 1991Co-Authors: Yoshihiro Ando, Sadao Imamura, M K Owada, Reiji KannagiAbstract:Abstract Covalently cross-linked multimers of Lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of Lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of Lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of Lipocortin I. Cross-linked Lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of Lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antiLipocortin-I antibody showed that cross-linked Lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified Lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked Lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using Lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of Lipocortin I. The cross-linking of Lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which Lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of Lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound Lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.