The mitochondrial respiratory chain from Rhodotorula mucilaginosa, an extremophile yeast.

Rhodotorula mucilaginosa survives extreme conditions through several mechanisms, among them its carotenoid production and its branched mitochondrial respiratory chain (RC). Here, the branched RC composition was analyzed by biochemical and complexome profiling approaches. Expression of the different RC components varied depending on the growth phase and the carbon source present in the medium. R. mucilaginosa RC is constituted by all four orthodox respiratory complexes (CI to CIV) plus several alternative oxidoreductases, in particular two type-II NADH dehydrogenases (NDH2) and one alternative oxidase (AOX). Unlike others, in this yeast the activities of the orthodox and alternative respiratory complexes decreased in the stationary phase. We propose that the branched RC adaptability is an important factor for survival in extreme environmental conditions; thus, contributing to the exceptional resilience of R. mucilaginosa.

Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis.

Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.

Bacteriostatic effect of Echeveria extracts on diarrheagenic E. coli pathotypes and non-cytotoxicity on human Caco-2 cells.

INTRODUCTION: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative. AIMS: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production. METHODOLOGY: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit. RESULTS: Extracts concentrations of 100 µg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells. CONCLUSIONS: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections.

Coupling/Uncoupling Reversibility in Isolated Mitochondria from Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae uses fermentation as the preferred pathway to obtain ATP and requires the respiratory chain to re-oxidize the NADH needed for activity of Glyceraldehyde-3-phosphate. This process is favored by uncoupling of oxidative phosphorylation (OxPhos), which is at least partially controlled by the mitochondrial unspecific pore (ScMUC). When mitochondrial ATP synthesis is needed as in the diauxic phase or during mating, a large rise in Ca2+ concentration ([Ca2+]) closes ScMUC, coupling OxPhos. In addition, ScMUC opening/closing is mediated by the ATP/ADP ratio, which indicates cellular energy needs. Here, opening and closing of ScMUC was evaluated in isolated mitochondria from S. cerevisiae at different incubation times and in the presence of different ATP/ADP ratios or varying [Ca2+]. Measurements of the rate of O2 consumption, mitochondrial swelling, transmembrane potential and ROS generation were conducted. It was observed that ScMUC opening was reversible, a high ATP/ADP ratio promoted opening and [Ca2+] closed ScMUC even after several minutes of incubation in the open state. In the absence of ATP synthesis, closure of ScMUC resulted in an increase in ROS.

Monitoring Mitochondrial Function in Aedes albopictus C6/36 Cell Line during Dengue Virus Infection.

Aedes aegypti and Aedes albopictus mosquitoes are responsible for dengue virus (DENV) transmission in tropical and subtropical areas worldwide, where an estimated 3 billion people live at risk of DENV exposure. DENV-infected individuals show symptoms ranging from sub-clinical or mild to hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The interactions between viruses and host mitochondria are crucial for virus replication and pathogenicity. DENV infection in vertebrate cells modulates mitochondrial function and dynamics to facilitate viral proliferation. Here, we describe that DENV also regulates mitochondrial function and morphology in infected C6/36 mosquito cells (derived from Aedes albopictus). Our results showed that DENV infection increased ROS (reactive oxygen species) production, modulated mitochondrial transmembrane potential and induced changes in mitochondrial respiration. Furthermore, we offer the first evidence that DENV causes translocation of mitofusins to mitochondria in the C6/36 mosquito cell line. Another protein Drp-1 (Dynamin-related protein 1) did not localize to mitochondria in DENV-infected cells. This observation therefore ruled out the possibility that the abovementioned alterations in mitochondrial function are associated with mitochondrial fission. In summary, this report provides some key insights into the virus-mitochondria crosstalk in DENV infected mosquito cells.

Integrative analysis of transcriptional profile reveals LINC00052 as a suppressor of breast cancer cell migration.

BACKGROUND: Long-non-coding RNAs, a class of transcripts with lengths > 200 nt, play key roles in tumour progression. Previous reports revealed that LINC00052 (long intergenic non-coding RNA 00052) was strongly downregulated during breast cancer multicellular spheroids formation and suggested a role in cell migration and oxidative metabolism. OBJECTIVE: To examine the function of LINC00052 in MCF-7 breast cancer cells. METHODS: Loss-of-function studies were performed to evaluate LINC00052 role on MCF-7 breast cancer cells. Microarray expression assays were performed to determine genes and cellular functions modified after LINC00052 knockdown. Next, the impact of LINC00052 depletion on MCF-7 cell respiration and migration was evaluated. RESULTS: 1,081 genes were differentially expressed upon LINC00052 inhibition. Gene set enrichment analysis, Gene Ontology and Key Pathway Advisor analysis showed that signalling networks related to cell migration and oxidative phosphorylation were enriched. However, whereas LINC00052 knockdown in MCF-7 cells revealed marginal difference in oxygen consumption rates when compared with control cells, LINC00052 inhibition enhanced cell migration in vitro and in vivo, as observed using a Zebrafish embryo xenotransplant model. CONCLUSION: Our data show that LINC00052 modulates MCF-7 cell migration. Genome-wide microarray experiments suggest that cancer cell migration is affected by LINC00052 through cytoskeleton modulation and Notch/ß-catenin/NF-κB signalling pathways.

Antimicrobial Peptide against Mycobacterium Tuberculosis That Activates Autophagy Is an Effective Treatment for Tuberculosis.

Mycobacterium tuberculosis (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 µM), while increasing the dose (50 µM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.

Age-Dependent Decline in Synaptic Mitochondrial Function Is Exacerbated in Vulnerable Brain Regions of Female 3xTg-AD Mice.

Synaptic aging has been associated with neuronal circuit dysfunction and cognitive decline. Reduced mitochondrial function may be an early event that compromises synaptic integrity and neurotransmission in vulnerable brain regions during physiological and pathological aging. Thus, we aimed to measure mitochondrial function in synapses from three brain regions at two different ages in the 3xTg-AD mouse model and in wild mice. We found that aging is the main factor associated with the decline in synaptic mitochondrial function, particularly in synapses isolated from the cerebellum. Accumulation of toxic compounds, such as tau and Aß, that occurred in the 3xTg-AD mouse model seemed to participate in the worsening of this decline in the hippocampus. The changes in synaptic bioenergetics were also associated with increased activation of the mitochondrial fission protein Drp1. These results suggest the presence of altered mechanisms of synaptic mitochondrial dynamics and their quality control during aging and in the 3xTg-AD mouse model; they also point to bioenergetic restoration as a useful therapeutic strategy to preserve synaptic function during aging and at the early stages of Alzheimer's disease (AD).

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei.

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions.

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.

Mitochondrial uncoupling proteins UCP4 and UCP5 from the Pacific white shrimp Litopenaeus vannamei.

Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954 bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3 kDa. The coding region of LvUCP5 transcript is 906 bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17 kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.

Wolbachia pipientis grows in Saccharomyces cerevisiae evoking early death of the host and deregulation of mitochondrial metabolism.

Wolbachia sp. has colonized over 70% of insect species, successfully manipulating host fertility, protein expression, lifespan, and metabolism. Understanding and engineering the biochemistry and physiology of Wolbachia holds great promise for insect vector-borne disease eradication. Wolbachia is cultured in cell lines, which have long duplication times and are difficult to manipulate and study. The yeast strain Saccharomyces cerevisiae W303 was used successfully as an artificial host for Wolbachia wAlbB. As compared to controls, infected yeast lost viability early, probably as a result of an abnormally high mitochondrial oxidative phosphorylation activity observed at late stages of growth. No respiratory chain proteins from Wolbachia were detected, while several Wolbachia F1 F0 -ATPase subunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.

Cholesterol burden in the liver induces mitochondrial dynamic changes and resistance to apoptosis.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of histopathological changes ranging from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis (NASH), which may progress into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. Recent data suggest that impaired hepatic cholesterol homeostasis and its accumulation are relevant to the pathogenesis of NAFLD/NASH. Despite a vital physiological function of cholesterol, mitochondrial dysfunction is an important consequence of dietary-induced hypercholesterolemia and was, subsequently, linked to many pathophysiological conditions. The aim in the current study was to evaluate the morphological and molecular changes of cholesterol overload in mouse liver and particularly, in mitochondria, induced by a high-cholesterol (HC) diet for one month. Histopathological studies revealed microvesicular hepatic steatosis and significantly elevated levels of liver cholesterol and triglycerides leading to impaired liver synthesis. Further, high levels of oxidative stress could be determined in liver tissue as well as primary hepatocyte culture. Transcriptomic changes induced by the HC diet involved disruption in key pathways related to cell death and oxidative stress as well as upregulation of genes related to glutathione homeostasis. Impaired liver function could be associated with a decrease in mitochondrial membrane potential and ATP content and significant alterations in mitochondrial dynamics. We demonstrate that cholesterol overload in the liver leads to mitochondrial changes which may render damaged hepatocytes proliferative and resistant to cell death whereby perpetuating liver damage.

Mitochondrial activity in different regions of the brain at the onset of streptozotocin-induced diabetes in rats.

Diabetes affects a variety of tissues including the central nervous system; moreover, some evidence indicates that memory and learning processes are disrupted. Also, oxidative stress triggers alterations in different tissues including the brain. Recent studies indicate mitochondria dysfunction is a pivotal factor for neuron damage. Therefore, we studied mitochondrial activity in three brain regions at early type I-diabetes induction. Isolated mitochondria from normal hippocampus, cortex and cerebellum revealed different rates of oxygen consumption, but similar respiratory controls. Oxygen consumption in basal state 4 significantly increased in the mitochondria from all three brain regions from diabetic rats. No relevant differences were observed in the activity of respiratory complexes, but hippocampal mitochondrial membrane potential was reduced. However, ATP content, mitochondrial cytochrome c, and protein levels of ß-tubulin III, synaptophysin, and glutamine synthase were similar in brain regions from normal and diabetic rats. In addition, no differences in total glutathione levels were observed between normal and diabetic rat brain regions. Our results indicated that different regions of the brain have specific metabolic responses. The changes in mitochondrial activity we observed at early diabetes induction did not appear to cause metabolic alterations, but they might appear at later stages. Longer-term streptozotocin treatment studies must be done to elucidate the impact of hyperglycemia in brain metabolism and the function of specific brain regions.

The role of glycolysis-derived hexose phosphates in the induction of the Crabtree effect.

Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect.

Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei.

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the F1FO-ATPase of Paracoccus denitrificans.

The biological roles of the three natural F1FO-ATPase inhibitors, ε, ζ, and IF1, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF1, but in the F1FO-ATPase of Paracoccus denitrificans (PdF1FO), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdF1FO-ATPase activity without affecting the PdF1FO-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdF1FO-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

Response of Ustilago maydis against the Stress Caused by Three Polycationic Chitin Derivatives.

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL-1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation.

During capacitation in bull spermatozoa, actin and PLC-ζ undergo dynamic interactions.

The migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the 'spermatozoidal activator factor' (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.

Key role of alternative oxidase in lovastatin solid-state fermentation.

Lovastatin is a commercially important secondary metabolite produced by Aspergillus terreus, either by solid-state fermentation or by submerged fermentation. In a previous work, we showed that reactive oxygen species (ROS) accumulation in idiophase positively regulates lovastatin biosynthetic genes. In addition, it has been found that lovastatin-specific production decreases with aeration in solid-state fermentation (SSF). To study this phenomenon, we determined ROS accumulation during lovastatin SSF, under high and low aeration conditions. Paradoxically, high aeration caused lower ROS accumulation, and this was the underlying reason of the aeration effect on lovastatin production. Looking for a mechanism that is lowering ROS production under those conditions, we studied alternative respiration. The alternative oxidase provides an alternative route for electrons passing through the electron transport chain to reduce oxygen. Here, we showed that an alternative oxidase (AOX) is expressed in SSF, and only during idiophase. It was shown that higher aeration induces higher alternative respiration (AOX activity), and this is a mechanism that limits ROS generation and keeps them within healthy limits and adequate signaling limits for lovastatin production. Indeed, the aox gene was induced in idiophase, i.e., at the time of ROS accumulation. Moreover, exogenous ROS (H2O2), added to lovastatin solid-state fermentation, induced higher AOX activity. This suggests that high O2 availability in SSF generates dangerously high ROS, so alternative respiration is induced in SSF, indirectly favoring lovastatin production. Conversely, alternative respiration was not detected in lovastatin-submerged fermentation (SmF), although exogenous ROS also induced relatively low AOX activity in SmF.