| 1. |
- Balbín, M, et al.
(författare)
-
Demonstration of sequence variations in the promoter region of the human cystatin C gene
- 1992
-
Ingår i: Biological Chemistry Hoppe-Seyler. - 0177-3593. ; 373:7, s. 471-476
-
Tidskriftsartikel (refereegranskat)abstract
- Four point mutations in the promoter region of the human cystatin C gene have been detected by direct sequencing of polymerase chain reaction (PCR) amplified DNA. The four base changes are all localized within a short segment of 85 base pairs. Three cystatin C gene alleles could be defined with respect to these promoter mutations; one with the sequence previously published, one carrying three of the mutations and one with all four base substitutions. Two of the observed mutations are involved in a novel Sst II polymorphism and another generates a new Dde I restriction site. A PCR-based assay for analysis of these Sst II and Dde I sites was designed and used to demonstrate Mendelian inheritance of the polymorphisms as well as to determine the frequencies of the cystatin C gene alleles in the population.
|
|
| 2. |
- Balbin, M, et al.
(författare)
-
Determination of allotypes G1m(f) and G1m(z) at the genomic level by subclass specific amplification of DNA and use of allele specific probes
- 1991
-
Ingår i: Experimental and Clinical Immunogenetics. - 0254-9670. ; 8:2, s. 88-95
-
Tidskriftsartikel (refereegranskat)abstract
- Two oligonucleotide primers were used for selective enzymatic amplification of a DNA segment encoding a major portion of the first constant region domain (CH1) of the human IgG1 heavy chain. The selective amplification was confirmed by use of subclass-specific oligonucleotide probes. Two 15-mer oligonucleotides, hybridizing with the alleles for the allotypes G1m(f) and (z), respectively, could then be used for determination at the genomic level of these two truly allelic allotypes. Serum and DNA samples from 12 individuals, one of them with a considerable amount of anti-Gm(f) antibodies, were used for allotype assignment by classical serological methods and by the new method operating at the genomic level. The resulting classifications agreed completely, demonstrating the reliability of the new method.
|
|
| 3. |
- Buttle, David J, et al.
(författare)
-
Human sputum cathepsin B degrades proteoglycan, is inhibited by a2-macroglobulin and modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C
- 1991
-
Ingår i: Biochemical Journal. - 0264-6021. ; 276, s. 325-331
-
Tidskriftsartikel (refereegranskat)abstract
- The high-Mr alkali-stable form of cathepsin B was purified from purulent human sputum. It was shown to solubilize proteoglycan monomer entrapped in polyacrylamide at a rate comparable with that of human lysosomal cathepsin B. Like the enzyme from lysosomes, sputum cathepsin B was bound by human alpha 2-macroglobulin, which inhibited its action on proteoglycan. Cystatin C in purulent sputum was shown to be the N-terminally truncated form generated by neutrophil elastase cleavage, and sputum cathepsin B was only weakly inhibited by recombinant cystatin C that had been cleaved by neutrophil elastase in vitro. Addition of neutrophil elastase to mucoid sputum led to a 5-fold increase in cathepsin B activity concomitant with a lowering in Mr of the cysteine proteinase from 40,000 to 37,000, i.e. the size of the active enzyme purified from purulent sputum. It is concluded that the high-Mr form of cathepsin B present in purulent sputum is a functional proteinase, unlike similar forms of the enzyme secreted by mammary gland in organ culture. The activity of cathepsin B in sputum is modulated by neutrophil elastase, by a combination of inhibitor inactivation and zymogen activation.
|
|
| 4. |
- Buttle, David J, et al.
(författare)
-
Interactions of papaya proteinase IV with inhibitors
- 1990
-
Ingår i: FEBS Letters. - 1873-3468. ; 262:1, s. 58-60
-
Tidskriftsartikel (refereegranskat)abstract
- Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by ‘papain’ reported previously. PPIV is slowly bound by human α2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.
|
|
