Characteristic transketolase alterations in dermal fibroblasts of Alzheimer patients are modulated by culture conditions.
Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline pI, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO2 saturation in the atmosphere and with 3.7 g/liter NaHCO3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.
Tombaccini
D
D
Istituto di Patologia Generale, Università di Firenze, Italy.
Mocali
A
A
Paoletti
F
F
D000544:Alzheimer Disease / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D002245:Carbon Dioxide / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D012867:Skin / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D014174:Transketolase / Q000032:analysis / Q000378:metabolism*
D002245:Carbon Dioxide; D014174:Transketolase