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| 2. |
- Abrahamson, Magnus, et al.
(författare)
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Efficient production of native, biologically active human cystatin C by Escherichia coli
- 1988
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Ingår i: FEBS Letters. - 1873-3468. ; 236:1, s. 14-18
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Tidskriftsartikel (refereegranskat)abstract
- A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the λ PR promoter, an optimized Shine-Dalgarno sequence and the λ cI 857 repressor. When induced at 42°C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.
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| 3. |
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| 4. |
- Abrahamson, Magnus
(författare)
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Human cysteine proteinase inhibitors. Isolation, physiological importance, inhibitory mechanism, gene structure and relation to hereditary cerebral hemorrhage
- 1988
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 48, s. 21-31
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Tidskriftsartikel (refereegranskat)abstract
- The isolation and characterization of six human cysteine proteinase inhibitors is reported. Their distribution in human biological fluids is also described and discussed with respect to physiological function. Studies on kininogen and cystatin C with respect to structure-function relationships and, as a result of the cystatin C studies, a general model for the mechanism of cysteine proteinase inhibition by cystatins are presented. The model was used for the construction of synthetic inhibitors which showed good inhibitory properties against papain and the streptococcal cysteine proteinase. Structures of cDNA and gene for normal human cystatin C are accounted for, as well as studies on the cystatin C gene in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). As a result of this an RFLP that showed total co-segregation with the disease was found. It was concluded that the disease is caused by a point mutation in the cystatin C structural gene and that the RFLP will be a most useful tool for diagnosis of HCCAA. The production of recombinant cystatin C in E. coli is also reported and its possible use for treatment of HCCAA is discussed.
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| 5. |
- Abrahamson, Magnus, et al.
(författare)
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Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin
- 1987
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 262:20, s. 9688-9694
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Tidskriftsartikel (refereegranskat)abstract
- When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy- trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate- like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.
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| 6. |
- Abrahamson, Magnus, et al.
(författare)
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Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids
- 1986
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 261:24, s. 11282-11289
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Tidskriftsartikel (refereegranskat)abstract
- Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
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| 7. |
- Abrahamson, Magnus, et al.
(författare)
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Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C
- 1987
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 216:2, s. 229-233
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant cystatin C producing clones were isolated from a human placenta λgt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.
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| 8. |
- Abrahamson, Magnus, et al.
(författare)
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The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20
- 1989
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Ingår i: Human Genetics. - 1432-1203. ; 82:3, s. 223-226
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Tidskriftsartikel (refereegranskat)abstract
- Hereditary cystatin C amyloid angiopathy has recently been shown to be caused by a point mutation in the cystatin C gene. To determine the chromosomal localization of the gene, 20 human-rodent somatic cell hybrids and a fulllength cystatin C cDNA probe were used. Southern blot analysis of BamHI digested cell hybrid DNA revealed that the probe recognizes a 10.6 kb human specific fragment and that this fragment cosegregates with human chromosome 20. Therefore, the human cystatin C gene (CST3) was assigned to chromosome 20.
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| 9. |
- Björck, Lars, et al.
(författare)
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Bacterial growth inhibited by a synthetic inhibitor based upon the structure of a human proteinase inhibitor
- 1989
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 337:6205, s. 385-386
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Tidskriftsartikel (refereegranskat)abstract
- Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins1 and in the processing of prohormones and proenzymes2,3, but also in the penetration of normal human tissue by malignant cells4 and possibly microorganisms5, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids6. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C7 and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracy-cline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.
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| 10. |
- Buttle, David, et al.
(författare)
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The biochemistry of the action of chymopapain in relief of sciatica
- 1986
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Ingår i: Spine. - 0362-2436. ; 11:7, s. 688-694
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Tidskriftsartikel (refereegranskat)abstract
- A study has been made of the mechanism of action of intradiscal injections of preparations of chymopapain in the treatment of sciatica. Such preparations were found to contain at least four distinct proteins, but enzymatically active chymopapain was the component mainly responsible for releasing glycosaminoglycan from cartilaginous tissue. Previous suggestions that an electrostatic interaction between chymopapain and glycosaminoglycan is important to the action of injected enzyme were not supported by the finding that both positively and negatively charged forms of chymopapain efficiently released glycosaminoglycan from cartilaginous tissue. In contrast, cysteine alone did not cause release of glycosaminoglycan. Chymopapain was found to be inhibited efficiently by the protein inhibitors, cystatin C and low molecular weight kininogen in vitro, and the possible relevance of this finding to the efficacy and safety of chemonucleolysis is discussed.
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