Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation oflarge T antigen causes p53 release, growth arrest, and cell death.Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (At,) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in A'I is associated with a decrease in the rate ofmitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.Mammalian cells grown in culture exhibit a finite life-span for proliferation. After a variable number of divisions they stop dividing, undergo a variety of changes, and finally die. Some oncogenes have the ability to confer an unlimited proliferative potential (immortality) to primary cells in culture. In the case of polyomavirus-induced immortalization of rodent embryo fibroblasts, it has been shown that the unlimited proliferative capacity is maintained by the large tumor (T) antigen (1)(2)(3)(4) MATERIALS AND METHODSCell Lines and Cell Culture. The REtsAF and RELPB cell lines were isolated at low cell density from a rat embryo fibroblast culture infected with SV40 (2). REtsAF was obtained by using a temperature-sensitive tsA58 mutant and is temperature sensitive for immortalization, whereas RELPB was obtained with wild-type SV40 and is immortal at both 330 and 39.50C. REtsAF-Revl was derived from REtsAF by selection for proliferation at 39.50C (8
Apoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-a treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-a-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-a-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-a-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondriadependent ROS production, the other being ROSindependent.
In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b, into its two functional domains: a cytochrome h, core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1 :< that of intact flavocytochrome b,. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome 6 , which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting thein is cleaved by Staphylococcus aureus V 8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80 "/, that of flavocytochrome b,. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of 'gene fusion'.Each chain of yeast flavocytochrome b, has been shown to be folded into three globules: n, E and / I ' joined together in the 'intact' enzyme by two protease-sensitive bridges aa' and cd [I -41 (cf. These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.This study presents information concerning this question. Despite the hypersensitivity of the aa' zone towards a number of proteases [I2 -141, we succeeded in obtaining a cleavage in the cd zone faster than in the aa' zone. This was achieved with Hansenula anomala flavocytochrome b2 and the highly specific Staphylococcus aureus V 8 protease 1. We describe a method for separating the so-formed 'intact' flavodehydrogenase part which we currently term 'FDH,,', from the other proteolysis products. Steady-state catalytic parameters of this new flavocytochrome h, derivative have been determined and compared with those of the native flavocytochrome b, and its tryptic derivatives. MATERIALS A N D METHODS Hansenula anomala Flavocytochrome b,This was prepared according to [I51 with the minor modifications described in [I I]. It was stored as an ammonium sulfate precipitate (50 % satur...
Laboratoire associe a I'Universite Pierre et Marie Curie, Gif-sur-Yvette The protomeric chain of Hansenula anomala flavocytochrome bz was previously shown to be built as the covalent association of two functional domains : an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H . anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer ( M , = 4 x 39000) containing FMN as expected and not heme. It has high L-1actate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same K,,, for L(+)-lactate as flavocytochrome hz, but it has no L-lactate: cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochromc hz. The subcellular origin of the H . anomala proteinase in the preparation has not yet been elucidated.Yeast flavocytochrome b, is a mitochondria1 enzyme located in the interinembrane space [I] and is involved in an electron pathway alternative to the main respiratory chain [2]. It was crystallized more than 30 years ago by Appleby and Morton [3] and has emerged as a model enzyme for the understanding of electron transfer either between a flavoprotein and a cytochrome or between two cytochromes (references in [4]). The mature enzyme is a tetramer [S] ( M , = 240000) 15 -81. Each protomer, in the native state, consists of a single polypeptide chain [7, 91 and
We carried out gain-of-function mutagenesis screening and identified a mutant in which GAL4 induction led to both hyperplasia and apoptosis. The gene involved was identified as stonewall (stwl), a myb-related gene involved in germ cell proliferation and differentiation during oogenesis. As observed with dmyb, the ectopic expression of stwl UY823 inhibited endoreplication in salivary glands. We also found that stwl UY823 overexpression, like overexpression of the wildtype gene, activated G1/S transition and apoptosis. The apoptosis triggered by stwl UY823 expression is correlated to induction of the proapoptotic gene reaper. Finally, the death of flies induced by ectopic stwl UY823 expression is efficiently prevented in vivo by triggering cell death in stwl UY823 -expressing cells. Our results suggest that stwl UY823 kills flies by causing inappropriate cell cycle entry, and that triggering the death of these overproliferating cells or slowing their proliferation restores viability.
The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, α and β, of the ‘cleaved’ form of the flavocytochrome b2 chain. These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2‐mercaptoethanol). They were then renatured in the presence of excesses of FMN and protoheme. The protoheme was found to be quantitatively bound to the α subunit, confirming previous findings. The flavin binds neither to α alone nor to β alone, but only to the reassociated αβ protomer. The results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L‐lactate cytochrome c reductase found specifically in yeasts.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.