Many biochemical, physiological and behavioural processes show circadian rhythms which are generated by an internal time-keeping mechanism referred to as the biological clock. According to rapidly developing models, the core oscillator driving this clock is composed of an autoregulatory transcription-(post) translation-based feedback loop involving a set of 'dock' genes. Molecular clocks do not oscillate with an exact 24-hour rhythmicity but are entrained to solar day/night rhythms by light. The mammalian proteins Cryl and Cry2, which are members of the family of plant blue-light receptors (cryptochromes) and photolyases, have been proposed as candidate light receptors for photoentrainment of the biological clock. Here we show that mice lacking the Cryl or Cry2 protein display accelerated and delayed free-running periodicity of locomotor activity, respectively. Strikingly, in the absence of both proteins, an instantaneous and complete loss of free-running rhythmicity is observed. This suggests that, in addition to a possible photoreceptor and antagonistic clock-adjusting function, both proteins are essential for the maintenance of circadian rhythmicity.

GATA-1 is a tissue-specific DNA-binding protein containing two zinc-finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non-transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D-dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E-dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus.

These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.

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Cumene hydroperoxide (Chp) and 4-hydroxynonenal (HNE) were used to investigate the effect of peroxidative challenge upon the glutathione (GSH) metabolism of human skin fibroblasts. Cellular GSH contents decreased during short-term incubations with Chp and oxidised glutathione (GSSG) was formed concomitantly. During longer incubations the GSH level was restored and the substrate flux through the pentose phosphate shunt increased. So in the presence of hydroperoxides the GSH level is maintained by reduction of GSSG.HNE caused a strong decrease in cellular GSH contents. Prolonged incubation with HNE lead to a rise in GSH contents above the basal level. The flux through the pentose phosphate shunt did not change during exposure to HNE. Hence, during incubation with HNE the cell maintains its GSH content by de novo synthesis of GSH. This conclusion is further substantiated by the findings with a cell strain deficient in GSH synthetase. These cells survived if incubated with Chp but not if exposed to HNE.GSH contents of normal cells from phase 11 (young) cultures and from phase I11 (aged) cultures responded similarly to Chp during short-term incubations and during a week of culture with the test compound. The flux through the pentose phosphate shunt rose much more in phase 111 than in phase I1 cells when incubated with the same concentration series of Chp. We conclude that during in vitro ageing the amount of NADPH needed to maintain cellular GSH levels in the presence of hydroperoxides increases, while the capacity to respond to such a challenge is not affected.The phenomenon of cells ageing in culture was first described by Hayflick and Moorhead [l] and has been reproduced in many different systems [2 -41. To explain this phenomenon two types of theory have been put forward: one invokes an underlying genetic programme [5] while the other presumes stochastically arising damage to be the basic cause. One of the main hypotheses of the second type is the freeradical theory of ageing [6]. Free-radical reactions, for instance those arising upon the one-electron reduction of oxygen 171, are presumed to damage the cell. One of the major effects of free-radical reactions in the cell is lipid peroxidation [8]. Cellular aerobic metabolism gives rise to several species of reactive oxygen, including superoxide, hydroperoxides and hydroxyl radicals [9, 101, against which the cell has defence mechanisms such as the glutathione system [ll]. During in vitro ageing the level of GSH remains almost constant [12, 131. While GSH is equally available in all cell organelles, glutathione reductase and glutathione peroxidase activities decrease considerably during in vitro ageing [13]. On the other Correspondence to M.