[Increase in cell metabolism in normal, diploid human glial cells in stationary cell cultures induced by meclofenoxate]. / Erhöhung von Zellstoffwechselleistungen in normalen, diploiden, menschlichen Glia-Zellen in stationären Zellkulturen induziert durch Meclofenoxat.

Quantitative biochemical studies were undertaken in order to examine the influence of the accumulation of lipofuscin in secondary lysosomes on cell metabolic activities of normal diploid human glia cells in a stationary cell culture system. Glia cells accumulate lipofuscin as a function of the duration of the stationary cultivation in vitro. The accumulation of lipofuscin can be decreased by the long-term treatment with the pharmacon meclofenoxate (centrophenoxine, Helfergin). Concomitant with the reduction of the accumulated lipofuscin, meclofenoxate-treated glia cells show enhanced rates of RNA synthesis, protein synthesis and glucose uptake. Most likely, in meclofenoxate-treated normal diploid human glia cells in vitro, the utilisation of glucose is shifted from glycolysis to the pentose phosphate pathway. The data suggest that the meclofenoxate-induced reduction of lipofuscin accumulation has a positive effect on cell metabolic functions and causes a delay of the cellular aging of the human glia cells in vitro.

Routes of flavodoxin and ferredoxin reduction in Escherichia coli. CoA-acylating pyruvate: flavodoxin and NADPH: flavodoxin oxidoreductases participating in the activation of pyruvate formate-lyase.

Flavodoxin and ferredoxin become reduced in Escherichia coli cells by oxidoreductase reactions which use pyruvate and NADPH as electron donor substrates. The two enzymes, which are minor proteins of this organism, were measured through the reduced flavodoxin-dependent activation of pyruvate formate-lyase. The NADPH-dependent enzyme, obtained homogeneously through Procion-red affinity chromatography, was identified as the flavoprotein 'component R' described previously by Fujii and Huennekens [J. Biol. Chem. 249, 6745-6753 (1974)]. The pyruvate-dependent enzyme was identified as CoA-acetylating pyruvate:flavodoxin (ferredoxin) oxidoreductase. Its catalytic properties in the forward, reverse, and the 14CO2-pyruvate exchange reaction are reported. The dihydro form of flavodoxin was characterized as the particular species involved in the activation of pyruvate formate-lyase. The activation process still occurs with 70% of maximal efficiency when the ratio [NADPH]/([NADP] + [NADPH]) is fixed at the intracellular 'anabolic reduction charge' value of 0.45, in conjunction with the NADPH-dependent enzyme. The [2Fe-2S] ferredoxin, though being readily used as electron acceptor of both oxidoreductases and having a redox potential similar to flavodoxin, proved incompetent in mediating the activation of pyruvate formate-lyase.

Expression of glial and vimentin type intermediate filaments in cultures derived from human glial material.

Several cultures established from biopsies of apparently normal adult human glial material showed no cells positive for glial fibrillary acidic protein (GFA) when examined after seven or more cumulative population doublings (CPD), although the established glioma line U251 MG showed approximately 3% GFA-positive cells, and U333 CG/343 MG clone 3 showed greater than 98% GFA-positive cells. Both the human glia delivered cultures and the glioma lines were positive when assayed with sera specific for vimentin. We therefore investigated the expression of GFA as a function of cumulative population doublings after the establishment of primary cultures. Under our experimental conditions, although GFA-positive cells were clearly present in the primary cultures accounting for some 3%-14% of the cells present, the GFA marker was subsequently lost, and the proliferating cultures expressed only the vimentin type of intermediate filament. Those cells that were GFA-positive also appeared to be vimentin-positive. GFA expression was not reinduced in cultures that had lost the GFA marker by treatment with dibutyryl cyclic AMP. We discuss two alternate hypotheses for the origin of the GFA-negative cells: (1) the cultures area of astrocyte origin but lost the ability to express GFA on culturing; (2) the cultures originate from cells of nonastrocyte origin present in the primary material.