| 1. |
- 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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| 2. |
- Abrahamson, Magnus
(författare)
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Molecular basis for amyloidosis related to hereditary brain hemorrhage
- 1996
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 56:226, s. 47-56
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.
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| 3. |
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| 4. |
- Grubb, A
(författare)
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Analysis of the immunochemical relationship between antigens by electrophoresis in agarose gels containing antibodies
- 1972
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - : Informa UK Limited. - 0085-591X .- 0036-5513 .- 1502-7686. ; 124, s. 59-65
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Tidskriftsartikel (refereegranskat)abstract
- A procedure is described, in which two antigens are brought to interact during their electrophoretic migration in an agarose gel containing antiserum. Three fundamental precipitation patterns can be discerned. These patterns correspond to the three basic precipitation patterns obtained with the double radial immunodiffusion procedure of Ouchterlony. The procedure described here can thus be used in the analysis of the immunochemical relationsship between antigens. It may also be used for estimating the percentage of cross-reacting antibodies in an antiserum. The procedure requires only a few hours.
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| 5. |
- Grubb, A, et al.
(författare)
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Human gamma-trace. Structure, function and clinical use of concentration measurements
- 1985
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 177, s. 7-13
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Tidskriftsartikel (refereegranskat)abstract
- The discovery, tissue distribution, concentration in extracellular fluids and structure of human gamma-trace are reported. The use of determinations of the cerebrospinal fluid concentration of gamma-trace in the diagnosis of hereditary cerebral hemorrhage with gamma-trace-amyloidosis is described. The physiological function of gamma-trace as a cysteine proteinase inhibitor is accounted for an it is suggested that the six trivial names used for gamma-trace so far are replaced by the functional designation cystatin C.
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| 6. |
- Hillarp, A
(författare)
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Molecular analysis of the beta-chain of human C4b-binding protein
- 1991
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - : Informa UK Limited. - 0085-591X .- 0036-5513 .- 1502-7686. ; 204, s. 57-69
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Forskningsöversikt (refereegranskat)abstract
- C4b-binding protein (C4BP) is a multimeric glycoprotein in plasma with important regulatory functions in the complement system. It occurs in two forms, as free protein and in a non-covalent bimolecular complex with the vitamin K-dependent protein S. Protein S is an important anticoagulant and enhances the rate of inactivation by activated protein C of blood coagulation factors, Va and VIIIa. Protein S bound to C4BP is inactive as an anticoagulant, indicating C4BP to have a regulatory function in the blood coagulation process. Approximately 50% of C4BP in plasma circulates in complex with protein S, but little has been known about as to how these proteins interact. This report describes the structure of C4BP and its relation to protein S binding. A novel C4BP subunit, designated the beta-chain, which in all likelihood contains the protein S binding site, has been identified, isolated and characterized. The major form of C4BP is composed of seven alpha-chains and one beta-chain, and the subunits are covalently linked by their carboxy-terminal regions giving the molecule a spider-like quaternary structure. A subpopulation of C4BP, which does not bind protein S, was found to lack the beta-chain. This provides support for the concept that the single protein S binding site is located on the beta-chain. The beta-chain is structurally related to the alpha-chain of C4BP, and both subunits belong to the superfamily of C3b/C4b-binding proteins. The genes coding for the alpha- and beta-chains of C4BP were found to be closely linked within a cluster of genes, coding for structurally related proteins, on the long arm of human chromosome 1.
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| 7. |
- Hokfelt, T, et al.
(författare)
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CCK-ergic mechanisms in sensory systems
- 2001
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 61234, s. 69-74
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Tidskriftsartikel (refereegranskat)
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| 8. |
- Ikonen, E, et al.
(författare)
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Dissection of the molecular pathology of aspartylglucosaminuria provides the basis for DNA diagnostics and future therapeutic interventions
- 1993
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Ingår i: Scandinavian journal of clinical and laboratory investigation. Supplementum. - 0085-591X. ; 213, s. 19-27
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Tidskriftsartikel (refereegranskat)abstract
- Aspartylglucosaminuria (AGU) is exceptional among lysosomal storage diseases since it represents the only known amidase deficiency in man, being caused by an inadequate function of aspartylglucosaminidase (AGA, E.C. 3.5.1.26.). This amidase is essential in one of the final steps in the ordered breakdown of glycoproteins since it cleaves Asn from the residual N-acetylglucosamines (for reviews see 1, 2). The deficiency of the enzyme activity results in the typical lysosomal accumulation of the abnormal degradation products (mainly aspartylglucosamine, 2-acetamido-1-beta-L-aspartamido-1,2-dideoxyglucose) in patients' cells and tissues. The diagnosis of AGU has so far been based on the detection of abnormal metabolites in urine and decreased enzyme activity in the cultured fibroblasts or isolated lymphocytes. Prenatal diagnosis has been possible by demonstrating the deficient enzyme activity of amniocytes or chorion villus biopsies. Identification of carriers has been difficult and unreliable due to the high individual variation in AGA activity and prerequisite for isolated blood lymphocytes. During the past few years we have purified the human enzyme into homogeneity, isolated the full length cDNA and characterized the majority of AGU mutations in this cDNA. This work facilitated the development of a reliable DNA diagnostic test suitable also for large scale carrier screening. The molecular pathology of the most common AGU mutation was unravelled, this being a prerequisite for the oncoming developments for therapy. Although AGU is a relatively rare disease, characterization of the AGU mutations and their cellular consequences have revealed highly interesting new phenomena in the biosynthesis of this lysosomal enzyme, some of which carry general biological significance.
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| 9. |
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| 10. |
- Lilja, H.
(författare)
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Regulation of the enzymatic activity of prostate-specific antigen and its reactions with extracellular protease inhibitors in prostate cancer
- 1995
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Ingår i: Scandinavian Journal of Clinical and Laboratory Investigation, Supplement. - 0085-591X. ; 55:220, s. 47-56
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Forskningsöversikt (refereegranskat)abstract
- Prostate-specific antigen (PSA) is a tissue-specific serine protease similar in structure to the trypsin-like glandular kallikreins but which is unique inasmuch as the enzyme activity is similar to that of chymotrypsin. The active enzyme is a single chain glycoprotein of 237 amino acids. The major form of PSA in serum is complexed to α1-antichymotrypsin (ACT). A small amount is free, non-complexed despite a large excess of ACT. This suggests that the form in serum lacks enzyme activity. Although serum PSA concentrations are regularly abnormally high (above 4 μg/L) in prostate cancer (CAP), the utility of PSA measurements in the early detection of CAP is limited, as many tumors are undetected at a cut-off of 4 μg/L. Also, 25% of all men with benign prostate hyperplasia (BPH) have serum PSA levels above 4 μg/L. Using assays specially developed to measure free and complexed forms of PSA in serum, we found the proportion of PSA-ACT complexes to be higher in CAP than in BPH, but the ratio of free-to-total PSA in serum to be lower. Using an abnormally low ratio of free-to-total PSA to detect CAP increases diagnostic specificity by 15 to 20%, compared to using a high serum PSA concentration. This suggests that the ratios of free-to-total PSA significantly increase the ability to distinguish BPH from localized CAP. The molecular basis is unclear, but may be related to the high incidence of prostate tumor cells producing both PSA and ACT. This is in contrast to the lack of ACT production in BPH epithelium. Possibly owing to lack of ACT production in BPH areas, conditions are not optimal for complex formation, whereas tumors producing both ACT and PSA may promote the formation of PSA-ACT complexes in CAP.
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