Abstract
A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy
Highlights
From the ‡Division of Oncology & Cellular Pathology, Pathology Section, University of Sheffield, Medical School, Beech Hill Road, Sheffield, S10 2RX, United Kingdom, the §Department of Rheumatology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom, the ʈNorthern Genetics Service, Newcastle upon Tyne, NE2 4AA, United Kingdom, and the ‡‡Department of Ophthalmology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, United Kingdom
S156C, G166C, and S181C s fundus dystrophy (SFD) Mutations—Mock-transfected COS-7 cells express 2 or 3 functional matrix metalloproteinases (MMPs) inhibitors in the extracellular matrix (ECM) with approximate molecular masses in the range of 23–29 kDa (Fig. 3, lane A). These masses correspond to those previously reported for unglycosylated and glycosylated tissue inhibitor of metalloproteinases-3 (TIMP-3), respectively [23], and the inhibitors are presumed to be endogenously expressed TIMP-3. This conclusion is confirmed by Western blotting (Fig. 4a, lane A) and by the fact that these bands are greatly intensified in COS-7 cells transfected with wild-type TIMP-3 (Figs. 3 and 4a, lane B)
We have identified an entirely novel TIMP-3 mutation in a family with Sorsby’s fundus dystrophy
Summary
DNA Samples—Blood was obtained from three affected members and one unaffected member of a recently identified SFD family, and genomic DNA was extracted by standard methods. DNA Sequencing—Polymerase chain reaction products amplified as above, from an unaffected control and an affected patient, were sequenced using SequenaseTM polymerase chain reaction product sequencing kit (Amersham Pharmacia Biotech) and the forward primer. For the Western blots, immediately after removing the cells and prior to harvesting the ECM, duplicate plates were incubated for 60 min at room temperature with 10 mM freshly prepared iodoacetamide to alkylate any free sulfhydryl groups. Immunoblotting—Reduced or nonreduced (heated for 10 min at 95 °C with or without 2% 2-mercaptoethanol respectively) samples were separated on 12% SDS-polyacrylamide gel electrophoresis gels with biotinylated molecular weight markers (Amersham Pharmacia Biotech) prior to blotting onto polyvinylidene difluoride membrane (Pierce & Warriner UK Ltd.) in 10 mM CAPS ϩ 10% methanol (pH 11.0) at 60mA overnight. Rabbit anti-TIMP-3 (first loop) antibody (Sigma) was diluted (1:1000) in TBS ϩ 6% skim milk powder ϩ 0.05% Tween-20 (antibody dilution buffer) and incubated with the blots overnight at 4 °C. Membranes were washed as before and developed with SuperSignal West Pico chemiluminescent substrate (Pierce)
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