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Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide., (© 2024 Federation of European Biochemical Societies.)

The point mutation S120G in human nucleoside diphosphate kinase A, identified in patients with neuroblastoma, causes a protein folding defect. The urea-unfolded protein cannot refold in vitro, and accumulates as a molten globule folding intermediate. We show here that the trimethylamine-N-oxide (TMAO) corrects the folding defect and stimulated subunit association. TMAO also substantially increased the stability to denaturation by urea of both wild-type and S120G mutant. A non-native folding intermediate accumulated in the presence of 4.5-7M urea and of 2M TMAO. It was inactive, monomeric, contained some secondary structure but no tertiary structure and displayed a remarkable stability to denaturation.

Macrophage ionotropic P2X7 receptors regulate cell-death through ill-defined signaling pathways. Here, we investigated the role of ceramide, an apoptogenic sphingolipid and showed that ATP stimulated ceramide accumulation in macrophages. Benzoylbenzoyl-ATP, a potent P2X7 agonist, was able to mimic the effects of ATP on ceramide accumulation while oxidized ATP had the opposite effect. Ceramide accumulation was blocked by de novo ceramide biosynthesis inhibitors. Interestingly, ATP-induced caspase-3/7 activation was dependent on ceramide generation. Finally, we showed that de novo ceramide biosynthesis is involved in ATP-induced macrophage death in a caspase-dependent manner. Our results indicate a novel role of ceramide in P2X7-regulated cell-death.

Rho proteins are down-regulated in vivo by specific GTPase activating proteins (RhoGAP). We have functionally studied three Saccharomyces cerevisiae putative RhoGAP. By first identifying Rho partners with a systematic two-hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p. In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac-like GTPase Rho5.

A new system for bax-expression in yeast has been devised to investigate bax's effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-xL.

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.

It has been suggested that the C-terminal domain of Bcl-2 family members may contain a signal anchor sequence that targets these proteins to the mitochondrial outer membrane. We have investigated the consequence of deleting this domain upon cytochrome c release in yeast strains that coexpress truncated forms of Bax (i.e. BaxA) and Bcl-X(L) (i.e. Bcl-X(L)delta). We find that (i) Bax(delta) is as efficient as full-length Bax in promoting cytochrome c release, but Bcl-x(L)delta has remarkably reduced rescuing ability compared to full-length Bcl-x(L); (ii) full-length Bcl-X(L) protein acts by relocalizing Bax from the mitochondrial fraction to the soluble cytosolic fraction; (iii) Bax undergoes N-terminal cleavage when expressed in yeast, which is prevented by coexpression of Bcl-X(L), suggesting that Bcl-x(L) may mask the cleavage site of Bax through a direct physical interaction of the two proteins.

The role of caspases in B lymphocyte cell death was investigated by using two broad spectrum inhibitors of the caspase family, Z-Asp-cmk and Z-VAD-fmk. They totally prevented spontaneous and drug-induced apoptosis and inhibited the CPP32/caspase-3-like activity exhibited by apoptotic cells. However, the suppression of apoptosis was not associated with a long-term increase of cell survival, but conversely, with a switch from apoptotic death to the necrotic form. These results strongly suggest that apoptosis and necrosis share common initiation pathways, the final issue being determined by the presence of an active caspase.

The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected. Coexpression of Bcl-xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.

By focusing on the question of the thermodynamic relationships involved in the regulation of biological energy conversion, bioenergetic studies usually consider the free pyridine and adenine nucleotide rather than their total pools, in either cytosol or mitochondria. In this study, we report a new observation that, at steady state, nicotinamide nucleotide content is increased by a rise in the ATP content of the whole cell under physiological conditions. It is a straight line relationship when only NAD+ and ATP are considered. When regarding the compartmentation of this phenomenon, it appears that the linear relationship between [NAD+] and [ATP] occurs only in the cytosol. Such a dependence could be a supplementary mechanism of regulation between various metabolic pathways in the liver cell.

Most if not all mitochondrial messenger RNAs from seed plants undergo a post-transcriptional modification (RNA editing) involving the conversion of some cytidine residues to uridine. Using a molecular hybridization approach, an in vitro RNA editing system, able to faithfully reproduce the in vivo observed C to U changes of subunit 9 (atp9) of wheat mitochondrial ATP synthase mRNA, has been described [Araya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1040-1044]. In this work we extend these studies to better understand the biochemical mechanism of this process. RNA editing was analysed by P1 nuclease digestion of the reaction product followed by thin layer chromatography. Experiments performed with unedited [3H]RNA labelled on the base and with unedited [32P]RNA labelled at the alpha-phosphate of cytidine residues, indicate that plant mitochondrial RNA editing operates through a deamination mechanism.

In the presence of KCl and only at low phosphate concentrations, ATP stimulated state 4 of the respiration of isolated yeast mitochondria. This effect could be related to a partial collapse of the transmembrane potential which was created by the respiratory chain or the F0F1-ATPase. Sodium and lithium could not replace potassium ion. Atractyloside prevented the opening of this K+ pathway, suggesting that only matricial ATP operated. All these effects were inhibited by increasing phosphate concentration, or by adding propranolol, quinine, Zn2+ or Mg2+.

The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms. The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture.

Retroviral RNA-dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion-encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNA(Lys3) is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNA(Lys.) By fluorescence studies and alpha-chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNA(Lys) although the retroviral polymerase is able to interact with other tRNAs.

Fast kinetic studies of cAMP accumulation in C6 cell membranes show a burst of cAMP after beta-adrenergic receptor stimulation by isoproterenol. This burst is no longer observed when the ATP present in membrane preparations is hydrolyzed, but can be restored by their preincubation in the presence of ATP-Mg. The size of the burst is much larger than the number of beta-adrenergic receptors and is of the same order of magnitude as the value reported for G proteins. Further characterization of the burst will allow studies of the functional interaction of receptor-adenylate cyclase components in C6 membranes.

The kinetics of changes in cytosolic free Ca2+ ([Ca2+]i) were determined in individual rat pancreatic acini by microfluorimetry. Three major findings are reported. First, at maximal stimulatory concentrations for amylase release, both caerulein and bombesin induced an initial rise in [Ca2+]i followed by prolonged secondary oscillations of smaller amplitude. The latter effect was not observed with supramaximal doses of caerulein. Second, these cyclic changes were dependent, at least in part, on extracellular Ca2+. Finally, comparison of the threshold doses for [Ca2+]i mobilization and enzyme discharge demonstrated that pathways independent of an elevation of [Ca2+]i control the secretory activity of pancreatic acini at low, picomolar agonist concentrations.

Bovine tRNA(Trp) can be partially hybridized to the avian myeloblastosis virus (AMV) 35 S RNA at 37 degrees C, in the presence of AMV RNA-dependent DNA polymerase (reverse transcriptase). This template-primer complex is active in the synthesis of viral cDNA. The size of the cDNA products synthesized in the in vitro reconstituted AMV system was determined by urea-polyacrylamide gel electrophoresis using a tRNA labelled at the 3'-end by yeast tRNA nucleotidyl transferase. The synthesized cDNA has a size of about 100 nucleotides and was shown by Southern blotting to be complementary to a specific sequence of the 5'-end of the retroviral genome. These results indicate that reverse transcriptase is able to anneal the exogenous primer tRNA at the 'primer-binding site' near the 5'-end of the long terminal repeat (LTR) of AMV RNA.

N(6)-2'-O-dibutyryl cyclic AMP stimulates glycogenolysis and inhibits fatty acid synthesis in rat liver in vivo but has little effect on gluconeogenesis. In adipose tissue: it enhances lipogenesis and glycogenesis. These last effects can be explained by an increase of insulin release.

The incorporations of 2,3-(14)C-succinate 2-(14)C-acetate into fatty acids of different cellular fractions of rat liver were studied. Acetate was incorporated mainly into supernatant and succinate into microsomal fatty acids. Mitochondria only could intensively decarboxylate pyruvate. Avidine inhibited fatty acid synthesis from succinate mainly in the supernatant. It is suggested that succinate is an important physiological precursor of fatty acids in the liver and that an active succino-dehydrogenase is present in microsomes.

The synthesis of fatty acids in mitochondria, microsomes and supernatant of rat adipose tissue was studied after administration of U-(14)C(-) glucose. The major site of fatty acids synthesis was the supernatant in controls and the microsomes in insulin-treated animals. The action of insulin cannot be explained only by an increase of the intra-cellular penetration of glucose. An action on enzyme synthesis is probable.

The repeating disaccharide unit of the mucopeptide of the cell wall of Mycobacterium smegmatis contains N-acetyl-glucosamine linked to N-glycolyl-muramic acid. This first identification of N-glycolyl-muramic acid as a natural product has been performed by mass spectrometry of the permethylated disaccharide. N-glycolyl muramic acid has been synthesized: the natural and synthetic products are identical.

The synthesis of fatty acids in mitochondria, microsomes and supernatant of mice liver was studied after administration of glucose 14C or (3)H, acetate 14C or 3H and pyruvate 14C.The results suggest that the cellular structures corresponding to the microsomes are the major site of fatty acid synthesis in the liver in vivo.

3',5'-Cyclic AMP inhibits fatty acid synthesis in mitochondria, microsomes and supernatant but does not provoke an enrichment in 3H with regard to 14C as it occurs during fasting after glucose 6-(3)H or 6-(14)C administration. Insulin does not exert any action on "controls" but restores lipogenesis in fasted animals in the examinated fractions and normalises the 3H/(14)C ratio, even that of microsomes. Control of neoglucogenesis by this hormone is more likely than by 3',5'-cyclic AMP.

2,4-(14) C-Citrate incorporated to a far greater extent than 1,5-(14) C-citrate into liver, carcass or adipose tissue fatty acids of living mice. This finding excludes the possibility that the acetyl units emerge from the mitochondria in the form of citrate.