Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.

GFP associates with microfilaments in fixed cells.

The discovery of the green fluorescent protein (GFP) and its use as a marker for proteins in cells revolutionised cell biology. Among its applications are the intracellular localisation of proteins and the investigation of the organisation, regulation and dynamics of the cytoskeleton. GFP itself is considered to be an inert protein, homogeneously distributed within the cytoplasm. Here we investigated the intracellular distribution of GFP in an amphibian and in various mammalian cell lines (XTH2, CHO-K1, HaCaT, MDCK, NIH-3T3) by confocal laser scanning microscopy. After paraformaldehyde fixation GFP became associated with microfilaments in all the cell lines investigated. This interaction was not impaired by detergent treatment (1% Brij 58 for 10 min). In contrast to the F-actin binding of GFP in fixed cells, association of GFP with stress fibres was not detectable in living cells. The actin-binding property of GFP might contribute also to the interaction of fusion proteins with microfilaments. Thus, careful controls are unavoidable in investigating (weak) actin-binding proteins in fixed cells. Because no association of GFP with microfilaments was detectable in living cells, it is recommended to monitor the intracellular distribution of GFP-tagged proteins in vivo.

Prevention of the acetylsalicylic acid-induced changes of the gastric potential difference by the new synthetic prostaglandin E1 analogue rioprostil.

The protective effect of 50 and 100 micrograms of the synthetic prostaglandin E1 analogue rioprostil (2-decarboxy-2-hydroxymethyl-15-deoxy-16RS-hydroxy-16-methyl PGE1) against the 0.5 and 1 g acetylsalicylic acid (ASA)-induced changes of gastric potential difference (PD) was compared to that of placebo in a double-blind cross-over study in 8 volunteers. Rioprostil 50 and 100 micrograms reduced significantly (p = 0.45) the intensity (area under baseline) of the 0.5 g ASA-induced PD changes. Both doses reduced highly significantly (p = 0.003) the time taken for PD to return to baseline value (15.1 min with 50 micrograms and 17.2 min with 100 micrograms rioprostil vs 43.5 min with placebo). Both doses of rioprostil also reduced significantly (p = 0.04) the PD maximal drop induced by 1 g ASA and reduced highly significantly (p = 0.006) the time taken for PD to return to baseline value (18.9 min with 50 micrograms and 16.8 min with 100 micrograms rioprostil vs 64.6 min with placebo). This study shows that rioprostil protects the stomach against the ASA-induced alterations of the gastric mucosa even when it is given at the same time as ASA and in doses which have only a small antisecretory effect.