The effects of delta-9-tetrahydrocannabinol administration in the regulation of female rat sociosexual behaviour.

By targeting the endocannabinoid system, delta-9-tetrahydrocannabinol (THC) modulates female motivated behaviours, influenced by sex hormones. Both medial preoptic nucleus (MPN) and ventromedial nucleus of the hypothalamus (VMN) are involved in the modulation of female sexual responses. The first triggers proceptivity, whereas the ventrolateral division of the latter (VMNvl) triggers receptivity. These nuclei are modulated by glutamate, which inhibits female receptivity, and GABA, which has a dichotomous action in female sexual motivation. Here, we evaluated the action of THC on the modulation of social and sexual behaviours, on signalling pathways of MPN and VMNvl and how sex hormones influence these parameters. Young ovariectomized female rats, given sex hormones (oestradiol benzoate, EB, and progesterone, P) and THC were used for behavioural testing and for immunofluorescence analyses of vesicular glutamate transporter 2 (VGlut2) and GAD (glutamic acid decarboxylase)67 expression. Results showed that females given EB + P exhibited a higher preference for male partner, as well as higher proceptivity and a higher receptivity than control or females given only EB. Females treated with THC presented similar responses in control or EB + P female rats and even more facilitated behavioural responses in EB females than the ones that did not receive THC. Immunofluorescence results in the MPN exhibited a decreased expression of GAD67 and VGlut2 in EB + THC-treated female rats. Within VMNvl of EB-primed rats no changes in the expression of both proteins were observed after THC exposure. This study demonstrates how the possible outcomes of endocannabinoid system instability within hypothalamic neuron connectivity can modify female rat sociosexual behaviour.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain­containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010 pg/mL and 2738 pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Protein Interactions in Rhodopseudomonas palustris TIE-1 Reveal the Molecular Basis for Resilient Photoferrotrophic Iron Oxidation.

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin-cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron-sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes.

Early unhealthy eating habits underlie morpho-functional changes in the liver and adipose tissue in male rats.

Early-life consumption of high-fat and sugar-rich foods is recognized as a major contributor for the onset of metabolic dysfunction and its related disorders, including diabetes and nonalcoholic fatty liver disease. The lifelong impact of early unhealthy eating habits that start at younger ages remains unclear. Therefore, to better understand the effects of diet, it is essential to evaluate the structural and functional changes induced in metabolic organs and potential mechanisms underlying those changes. To investigate the long-term effects of eating habits, young male rats were exposed to high-sugar and high-energy diets. After 14 weeks, body composition was assessed, and histopathological changes were analyzed in the liver and adipose tissue. Serum biochemical parameters were also determined. Expression of inflammatory markers in the liver was evaluated by immunohistochemistry. Our results revealed that serum levels of glucose, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), and lipid profile were increased in rats red high-sugar and high-energy diets. Histopathological alterations were observed, including abnormal hepatocyte organization and lipid droplet accumulation in the liver, and abnormal structure of adipocytes. In both unhealthy diet groups, hepatic expression of Toll-like receptor 4 (TLR4), cyclooxygenase 2 (COX-2), and E-selectin were increased, as well as a biomarker of oxidative stress. Together, our data demonstrated that unhealthy diets induced functional and structural changes in the metabolic organs, suggesting that proinflammatory and oxidative stress mechanisms trigger the hepatic alterations and metabolic dysfunction.

The role of macrophages phenotypes in the activation of resolution pathways within human granulosa cells.

BACKGROUND: Inflammatory state within the ovaries can disrupt normal follicular dynamics, leading to reduced oocyte quality and infertility. How the production of inflammatory mediators generated by macrophages with different gene expression profile (M1 and M2) might activate inflammatory pathways, such as cyclooxygenase-2 (COX-2) and 5-, 12-, and 15-lipoxygenase (LOX), in human granulosa cells (hGCs) remains unclear. METHODS: In this study, we evaluated how M1 and M2 macrophages found in the ovaries affect the functions of hGCs isolated from women undergoing assisted reproductive technology (ART) and human ovarian granulosa COV434 cells. For this purpose, a model of interaction between hGCs and COV434 cells and conditioned media (CMs) obtained from culture of M0, M1 and M2 macrophages was established. We used real-time PCR and western blotting to detect the expression of COX-2 and 5-, 12-, and 15-LOX as biomarkers of oocyte competence. RESULTS: Our data showed that M2 macrophages with anti-inflammatory characteristics were able to significantly increase the expression of COX-2 in hGCs. We also demonstrated that M1 macrophages with pro-inflammatory characteristics were able to significantly increase the expression of 12-LOX in hGCs. However, there was no observed expression of 5-LOX and no significant alteration in the expression of 15-LOX in hGCs. Regarding COV434 cells, we found that CM from M2 macrophage resulted in an increase in COX-2, 5-LOX and 15-LOX mRNA and protein levels. No expression of 12-LOX by COV434 cells was observed when exposed to CMs from M1 and M2 macrophages. CONCLUSIONS: Our research indicated that the production of pro-resolving mediators by hGCs can, at least in part, reverse the physiological inflammation present in the ovaries.

Unhealthy Diets Induce Distinct and Regional Effects on Intestinal Inflammatory Signalling Pathways and Long-Lasting Metabolic Dysfunction in Rats.

The intestinal epithelium is a principal site for environmental agents' detection. Several inflammation- and stress-related signalling pathways have been identified as key players in these processes. However, it is still unclear how the chronic intake of inadequate nutrients triggers inflammatory signalling pathways in different intestinal regions. We aimed to evaluate the impact of unhealthy dietary patterns, starting at a younger age, and the association with metabolic dysfunction, intestinal inflammatory response, and obesity in adulthood. A rat model was used to evaluate the effects of the consumption of sugary beverages (HSD) and a Western diet (WD), composed of ultra-processed foods. Both diets showed a positive correlation with adiposity index, but a positive correlation was found between the HSD diet and the levels of blood glucose and triglycerides, whereas the WD diet correlated positively with triglyceride levels. Moreover, a distinct inflammatory response was associated with either the WD or HSD diets. The WD induced an increase in TLR2, TLR4, and nuclear factor-kappa B (NF-κB) intestinal gene expression, with higher levels in the colon and overexpression of the inducible nitric oxide synthase. In turn, the HSD diet induced activation of the TLR2-mediated NF-κB signalling pathway in the small intestine. Altogether, these findings support the concept that early intake of unhealthy foods and nutrients are a main exogenous signal for disturbances of intestinal immune mechanisms and in a region-specific manner, ultimately leading to obesity-related disorders in later life.

Concentrations of the endocannabinoid N-arachidonoylethanolamine in the follicular fluid of women with endometriosis: the role of M1 polarised macrophages.

Although N-arachidonoylethanolamine (AEA; also known as anandamide) is present in human follicular fluid (FF), its regulation remains unknown. Therefore, the aims of the present study were to: (1) investigate the relationships between FF AEA concentrations in women undergoing assisted reproductive technology and their age, body mass index, ART characteristics and fertility treatment outcomes; and (2) assess how different inflammatory patterns may trigger AEA production by human granulosa cells (hGCs). FF AEA concentrations were higher in women undergoing IVF than in those undergoing intracytoplasmic sperm injection group. FF AEA median concentrations were lower in women undergoing ART because of male factor infertility than in women with endometriosis (1.6 vs 2.5nM respectively), but not women with tubal, hormonal or unexplained infertility (1.6, 2.4 and 1.9nM respectively). To evaluate the effects of macrophages on AEA production by hGCs, hGCs were cocultured with monocyte-derived macrophages. The conditioned medium from M1 polarised macrophages increased AEA production by hGCs. This was accompanied by an increase in AEA-metabolising enzymes, particularly N-acyl phosphatidylethanolamine-specific phospholipase D. The results of the present study show that high FF AEA concentrations in patients with endometriosis may be associated with the recruitment of inflammatory chemokines within the ovary, which together may contribute to the decreased reproductive potential of women with endometriosis. Collectively, these findings add a new player to the hormone and cytokine networks that regulate fertility in women.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility.

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.

Uterine histopathological changes induced by acute administration of tamoxifen and its modulation by sex steroid hormones.

The endometrium is a particular sensitive target tissue for estradiol that is able to promptly modify its structure. Tamoxifen (TAM), a selective estrogen receptor modulator, was shown to promote a spectrum of uterine abnormalities, though the morphological and stereological effects of this drug in uterus is not clear. In this way, we have used an established model of ovariectomy followed by estradiol benzoate (EB) or TAM treatment and analyzed their effects in uterine histopathology and proliferation. Administration of EB promotes the unfolding and proliferation of the endometrium stroma, increasing uterine volume. No changes were found in uterine histomorphometric analysis upon TAM administration, except in the thickness of the luminal epithelium and endometrium layer. The latter may result from increased complexity and glandular volume density also observed in TAM treatment. In addition, EB induced PAX2 expression, an oncogene commonly found in epithelial tumors of the female genital tract, an effect that was weakened by previous TAM administration. Although treatments did not affect stroma cells proliferating index, in epithelial cells and, contrary to TAM, EB increased PCNA and not Ki67 expression. Collectively, our data suggest that the acute administration of TAM induces ERα-dependent atrophy of the uterine tissue and decreased the expression of proliferating cellular markers. On the contrary, if administered prior to EB, TAM is able to attenuate the action of the hormone in uterine morphology and biochemistry.

Effects of tamoxifen on neuronal morphology, connectivity and biochemistry of hypothalamic ventromedial neurons: Impact on the modulators of sexual behavior.

Tamoxifen (TAM) is a selective estrogen receptor modulator, widely used in the treatment and prevention of estrogen-dependent breast cancer. Although with great clinical results, women on TAM therapy still report several side effects, such as sexual dysfunction, which impairs quality of life. The anatomo-functional substrates of the human sexual behavior are still unknown; however, these same substrates are very well characterized in the rodent female sexual behavior, which has advantage of being a very simple reflexive response, dependent on the activation of estrogen receptors (ERs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl). In fact, in the female rodent, the sexual behavior is triggered by increasing circulation levels of estradiol that changes the nucleus neurochemistry and modulates its intricate neuronal network. Therefore, we considered of notice the examination of the possible neurochemical alterations and the synaptic plasticity impairment in VMNvl neurons of estradiol-primed female rats treated with TAM that may be in the basis of this neurological disorder. Accordingly, we used stereological and biochemical methods to study the action of TAM in axospinous and axodendritic synaptic plasticity and on ER expression. The administration of TAM changed the VMNvl neurochemistry by reducing ERα mRNA and increasing ERß mRNA expression. Furthermore, present results show that TAM induced neuronal atrophy and reduced synaptic connectivity, favoring electrical inactivity. These data suggest that these cellular and molecular changes may be a possible neuronal mechanism of TAM action in the disruption of the VMNvl network, leading to the development of behavioral disorders.

Cannabinoid-induced autophagy: Protective or death role?

Autophagy, the "self-digestion" mechanism of the cells, is an evolutionary conserved catabolic process that targets portions of cytoplasm, damaged organelles and proteins for lysosomal degradation, which plays a crucial role in development and disease. Cannabinoids are active compounds of Cannabis sativa and the most prevalent psychoactive substance is Δ(9)-tetrahydrocannabinol (THC). Cannabinoid compounds can be divided in three types: the plant-derived natural products (phytocannabinoids), the cannabinoids produced endogenously (endocannabinoids) and the synthesized compounds (synthetic cannabinoids). Various studies reported a cannabinoid-induced autophagy mechanism in cancer and non-cancer cells. In this review we focus on the recent advances in the cannabinoid-induced autophagy and highlight the molecular mechanisms involved in these processes.

Lipidomic approach towards deciphering anandamide effects in rat decidual cell.

Altered phospholipid (PL) metabolism has been associated with pregnancy disorders. Moreover, lipid molecules such as endocannabinoids (eCBs) and prostaglandins (PGs) are important mediators of reproductive events. In humans, abnormal decidualization has been linked with unexplained infertility, miscarriage and endometrial pathologies. Anandamide (AEA), the major eCB, induces apoptosis in rat decidual cells. In this study, the PL profile of rat decidual cells was characterized by a Mass spectrometry (MS) based lipidomic approach. Furthermore, we analyzed a possible correlation between changes in PL of rat decidual cells' membrane and AEA-induced apoptosis. We found an increase in phosphatidylserine and a reduction of cardiolipin and phophatidylinositol relative contents. In addition, we observed an increase in the content of alkyl(alkenyl) acylPL, plasmalogens, and of long chain fatty acids especially with high degrees of unsaturation, as well as an increase in lipid hydroperoxides in treated cells. These findings provide novel insights on deregulation of lipid metabolism by anandamide, which may display further implications in decidualization process.

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1.

Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein-protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.

Alterations in the pro-resolving lipid mediator machinery within first trimester maternal tissue: Implications in decidualization and miscarriage risk.

A pivotal event in uterine receptivity and human reproduction is the differentiation of endometrial stromal cells into decidual cells, known as decidualization. Decidualization is interlinked with its inflammatory environment. Our study aimed to investigate the presence and role of pro-resolving lipid mediators in first trimester maternal tissue. We assessed the levels of LXA4 and RvD1, along with their metabolic LOX enzymes, in elective (control) and sporadic miscarriage samples. We investigated the effects of LXA4 and RvD1 on decidualization using primary endometrial stromal cells and the immortalized endometrial stromal St-T1b cell line. The upregulation of 12- and 15-LOX expression was observed in pregnancy tissue after sporadic miscarriage, suggesting an inflammatory imbalance. Furthermore, incubation with these lipid mediators led to a decrease in decidualization biomarkers PRL and IGFBP-1, accompanied by morphological changes indicative of aberrant differentiation. The expression of LOX enzymes in decidual natural killer cells suggests their involvement in regulating the inflammatory surroundings and the extent of decidualization.

Retinoic acid (all-trans) presents antioxidant properties within human ovary and reduces progesterone production by human granulosa cells.

Both vitamin A and E support female reproduction and embryonic development. These vitamins have been associated with decreased fertility or failure to end the pregnancy in animals. An observational study was conducted on follicular fluid (FF) samples to determine the concentrations of fat-soluble vitamins of women undergoing in vitro fertilization and its correlation with assisted reproductive technology characteristics and pregnancy outcomes. Moreover, the effects of all-trans-retinoic acid (atRA) and alpha-tocopherol on granulosa cell viability, apoptosis, autophagy and hormonal production were evaluated. No association was identified between fat-soluble vitamin concentrations in FF and infertility aetiology, body mass index or woman's age. There were differences in follicular antioxidant profiles and ovarian response stimulation. In vitro evaluation of atRA and alpha-tocopherol reveals that, at physiological concentrations, both compounds may affect the viability of granulosa cells. In addition, these compounds are able to protect granulosa cells from oxidative stress, as well as to affect estradiol and progesterone production. Our data suggest that atRA and alpha-tocopherol levels should be well controlled as they may have implications in the function and viability of granulosa cells and highlights retinol as a marker of the oxidative defenses within ovary environment.

Long-Term Tamoxifen Effects in the Cyclic Interaction of the Endocannabinoid and Endocrine System in the Rat Central Nervous System.

Steroid hormones can modulate the endocannabinoid system (ECS). Within the female reproductive tract, estrogen increases the expression of the cannabinoid receptors CB1 and CB2, and modifies the levels of anandamide (AEA), the major endocannabinoid, by altering the expression of both AEA synthesis (NAPE-PLD) and catabolic enzymes (FAAH). Here, we addressed the mechanisms involved in ECS fluctuations within the central nervous system and evaluated the effects of tamoxifen (TAM), a selective estrogen receptor modulator, in central AEA regulation. The current results suggest that the hypothalamic and pituitary AEA levels change differently according to the brain area and phase of the estrous cycle. In TAM-treated rats, there is a disruption of the cyclic fluctuation and reduction of the AEA levels in all brain areas. In the pituitary gland, NAPE-PLD expression increases in the metestrus phase, whereas throughout the rat cycle their expression remained constant, even upon TAM treatment. The fluctuations of pituitary AEA levels result from altered FAAH and NAPE-LPD expression. In contrast, no differences in FAAH or NAPE-PLD hypothalamic expression were observed. Overall, this study presents a broad view of the distribution and expression of ECS elements in the central nervous system and a way to suggest possible brain areas involved in the interaction of the endocannabinoid and neuroendocrine systems to induce several behavioral responses.

Cannabis and Cannabinoids in Reproduction and Fertility: Where We Stand.

Although cannabis use is increasing in general population, their prevalence among young adults is remarkably high. In recent years, their medical use gained a renewed interest. However, it can underline the reputation of cannabis being a harmless drug. Between cannabinoids, uniquely found on the cannabis plant, Δ9-tetrahydrocannabinol (THC) is the well-studied compound. It is responsible for the psychoactive effects via central cannabinoid receptors. Nevertheless, cannabinoids interact with other chemical signalling systems such as the hypothalamic-pituitary-gonadal axis. THC indirectly decreases gonadotropin-releasing hormone (GnRH) secretion by the hypothalamus. The consequences are diverse, and several key hormones are affected. THC disturbs important reproductive events like folliculogenesis, ovulation and sperm maturation and function. Although generally accepted that cannabinoid consumption impacts male and female fertility, prevailing evidence remains largely on pre-clinical studies. Here, we introduce cannabinoids and the endocannabinoid system, and we review the most prominent clinical evidence about cannabis consumption in reproductive potential and teratogenicity.

Effects of chronic tamoxifen treatment in female rat sexual behaviour.

The medial preoptic (MPN) and the ventromedial hypothalamic nuclei (VMN) modulate the estrogen receptor (ER)-dependent female sexual behavior, a response that is inhibited by tamoxifen (TAM), a modulator of the steroid receptor activation. With the objective to assess TAM action in the brain areas involved in the modulation sexual cues, an animal model on long-term TAM therapy to intact female rats, was used to mimic the 5-year prophylactic TAM therapy offered to women at higher risk of breast cancer. After three months treatment, female sexual behavior with a stud male rat was evaluated. Upon sacrifice, the brains were removed and the MPN and the ventrolateral division of the VMN were screened for the effects of TAM in the expression of ERα, ERß and progesterone receptor. Results show that TAM inhibited the receptive component of the female sexual behavior. Even though TAM decreased estrogen and progesterone levels to values similar to the ones of estrous and diestrus rats, the biochemical data failed to demonstrate such possible causation for the behavioral response. In fact, TAM administration induced a constant low level of ovarian hormones that changed the pattern of ER and PR expression as well as receptor co-expression in the brain areas regulating the behavioral response, dissimilar to the ones seen in the cycle phases with the same low hormone levels. Nevertheless, present data suggests that by affecting ER- and/or PR-dependent mechanisms, TAM may modulate the hypothalamus, a region known to participate in several social behaviors.

Deciphering Molecular Factors That Affect Electron Transfer at the Cell Surface of Electroactive Bacteria: The Case of OmcA from Shewanella oneidensis MR-1.

Multiheme cytochromes play a central role in extracellular electron transfer, a process that allows microorganisms to sustain their metabolism with external electron acceptors or donors. In Shewanella oneidensis MR-1, the decaheme cytochromes OmcA and MtrC show functional specificity for interaction with soluble and insoluble redox partners. In this work, the capacity of extracellular electron transfer by mutant variants of S. oneidensis MR-1 OmcA was investigated. The results show that amino acid mutations can affect protein stability and alter the redox properties of the protein, without affecting the ability to perform extracellular electron transfer to methyl orange dye or a poised electrode. The results also show that there is a good correlation between the reduction of the dye and the current generated at the electrode for most but not all mutants. This observation opens the door for investigations of the molecular mechanisms of interaction with different electron acceptors to tailor these surface exposed cytochromes towards specific bio-based applications.

In Vitro Effects of Mitochondria-Targeted Antioxidants in a Small-Cell Carcinoma of the Ovary of Hypercalcemic Type and in Type 1 and Type 2 Endometrial Cancer.

Small-cell carcinoma of the ovary of hypercalcemic type (SCCOHT) and endometrial cancer from type 1 and type 2 are gynecological tumors that affect women worldwide. The treatment encompasses the use of cytotoxic drugs that are nonspecific and inefficient. "Mitocans", a family of drugs that specifically target tumor cells' mitochondria, might be a solution, as they conjugate compounds, such as antioxidants, with carriers, such as lipophilic cations, that direct them to the mitochondria. In this study, caffeic acid was conjugated with triphenylphosphonium (TPP), 4-picolinium, or isoquinolinium, forming 3 new compounds (Mito6_TPP, Mito6_picol., and Mito6_isoq.) that were tested on ovarian (COV434) and endometrial (Hec50co and Ishikawa) cancer cells. The results of MTT and neutral red assays suggested a time- and concentration-dependent decrease in cell viability in all tumor cell lines. The presence of apoptosis was indicated by the Giemsa and Höechst staining and by the decrease in mitochondrial membrane potential. The measurement of intracellular reactive oxygen species demonstrated the antioxidant properties of these compounds, which might be related to cell death. Generally, Mito6_TPP was more active at lower concentrations than Mito6_picol. or Mito6_isoq., but was accompanied by more cytotoxic effects, as shown by the lactate dehydrogenase release. Non-tumorous cells (HFF-1) showed no changes after treatment. This study assessed the potential of these compounds as anticancer agents, although further investigation is needed.