Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, P-32-poly(Glu-Tyr)(4:1) and P-32-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria

Effects of essential oils from Cymbopogon spp. and Cinnamomum verum on biofilm and virulence properties of Escherichia coli O157:H7

Every year, the pharmaceutical and food industries produce over 1000 tons of essential oils (EOs) exploitable in different fields as the development of eco-friendly and safe antimicrobial inhibitors. In this work we investigated the potential of some EOs, namely Cinnamomum verum, Cymbopogon martini, Cymbopogoncitratus and Cymbopogon flexuosus, on the growth, biofilm formation and gene expression in four strains of enterohemorrhagic Escherichia coli O157:H7. All EOs were analyzed by gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity was performed by using dilutions of EOs ranging from 0.001 to 1.2% (v/v). Subinhibitory doses were used for biofilm inhibition assay. The expression profiles were obtained by RT-PCR. E. coli O157:H7 virulence was evaluated in vivo in the nematode Caenorhabditis elegans. All EOs showed minimal inhibitory concentrations (MICs) ranging from 0.0075 to 0.3% (v/v). Cinnamomum verum bark EO had the best activity (MIC of 0.0075% (v/v) in all strains) while the C. verum leaf EO had an intermediate efficacy with MIC of 0.175% (v/v) in almost all strains. The Cymbopogon spp. showed the more variable MICs (ranging from 0.075 to 0.3% (v/v)) depending on the strain used. Transcriptional analysis showed that C. martini EO repressed several genes involved in biofilm formation, virulence, zinc homeostasis and encoding some membrane proteins. All EOs affected zinc homeostasis, reducing ykgM and zinT expression, and reduced the ability of E. coli O157:H7 to infect the nematode C. elegans. In conclusion, we demonstrated that these EOs, affecting E. coli O157:H7 infectivity, have a great potential to be used against infections caused by microorganisms

Inositols: From established knowledge to novel approaches

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects

When one size does not fit all: Reconsidering PCOS etiology, diagnosis, clinical subgroups, and subgroup-specific treatments

Polycystic Ovary Syndrome (PCOS) is a complex endocrine disorder that affects a large proportion of women. Due to its heterogeneity, the best diagnostic strategy has been a matter of contention. Since 1990 scientific societies in the field of human reproduction have tried to define the pivotal criteria for the diagnosis of PCOS. The consensus Rotterdam diagnostic criteria included the presence of hyperandrogenism, oligo/anovulation, and polycystic ovarian morphology (PCOM), and have now been updated to evidence based diagnostic criteria in the 2018 and 2023 International Guideline diagnostic criteria endorsed by 39 societies internationally. Within the Rotterdam Criteria, at least two out of three of the above-mentioned features are required to be present to diagnose PCOS, resulting in four phenotypes being identified: phenotype A, characterized by the presence of all the features, phenotype B, exhibiting hyperandrogenism and oligo-anovulation, phenotype C, presenting as hyperandrogenism and PCOM and finally the phenotype D that is characterized by oligo-anovulation and PCOM, lacking the hyperandrogenic component. However, it is the hypothesis of the EGOI group that the Rotterdam phenotypes A, B, and C have a different underlying causality to phenotype D. Recent studies have highlighted the strong correlation between insulin resistance and hyperandrogenism, and the pivotal role of these factors in driving ovarian alterations, such as oligo-anovulation and follicular functional cyst formation. This new understanding of PCOS pathogenesis has led the authors to hypothesis that phenotypes A, B, and C are endocrine-metabolic syndromes with a metabolic clinical onset. Conversely, the absence of hyperandrogenism and metabolic disturbances in phenotype D suggests a different origin of this condition, and point towards novel pathophysiological mechanisms; however, these are still not fully understood. Further questions have been raised regarding the suitability of the “phenotypes” described by the Rotterdam Criteria by the publication by recent GWAS studies, which demonstrated that these phenotypes should be considered clinical subtypes as they are not reflected in the genetic picture. Hence, by capturing the heterogeneity of this complex disorder, current diagnostic criteria may benefit from a reassessment and the evaluation of additional parameters such as insulin resistance and endometrial thickness, with the purpose of not only improving their diagnostic accuracy but also of assigning an appropriate and personalized treatment. In this framework, the present overview aims to analyze the diagnostic criteria currently recognized by the scientific community and assess the suitability of their application in clinical practice in light of the newly emerging evidence

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model

Influence of the Morphology of Lysozyme-Shelled Microparticles on the Cellular Association, Uptake, and Degradation in Human Breast Adenocarcinoma Cells.

he ultrasound-assisted self-assembly and cross-linking of lysozyme at the water–air and water–perfluorohexane interfaces are shown to produce lysozyme-shelled hollow microbubbles (LSMBs) and microcapsules (LSMC), respectively. The arrangement of lysozyme at the air–liquid or oil–liquid interfaces is accompanied by changes in the bioactivity and conformational state of the protein. The interaction of LSMB and LSMC with human breast adenocarcinoma cells (SKBR3) is studied. LSMB and LSMC are phagocyted by cells within 2 h without exerting a cytotoxic activity. The cellular internalization kinetics of LSMB and LSMC and the effects on cell cycle are evaluated using flow cytometry. Evidence for the internalization of microparticles and degradation within the cell are also monitored by confocal and scanning electron microscopic analyses. The integrity of cell membrane and cell cycle is not affected by LSMBs and LSMCs uptake. These studies show that the positively charged LSMB and LSMC are not cytotoxic and can be readily internalized and degraded by the SKBR3 cells. LSMBs and LSMCs show a different uptake kinetics and intracellular degradation pattern due to differences in the arrangement of the protein at the air–liquid or oil–liquid interfaces

The Vepris macrophylla essential oil produces notable antiproliferative activity and morphological alterations in human breast adenocarcinoma cells

Medicinal plants contain numerous bioactive molecules that synergistically provide therapeutic benefits. We have devoted our attention to various EOs without toxicity to normal cells, studying their activities against human cancer cells. In particular, we have studied the cytotoxicity of Vepris macrophylla (Baker) I. Verd. EO. V. macrophylla is an evergreen tree of Madagascar where is much appreciated as a source of traditional remedies. Its major volatile components are citral, i.e., a mixture of neral and geranial, citronellol and myrcene. The antiproliferative activities of V. macrophylla EO were studied against human breast adenocarcinoma cell line SKBR3. Cellular metabolism was analyzed by MTT assay at different concentrations of EO and at different times of incubation (24, 48 and 72 h). Moreover, morphological and ultrastructural analyses were performed to study its antiproliferative effects against human adenocarcinoma cells, demonstrating the ability of V. macrophylla EO, stored inside numerous intracellular vesicles, to damage both plasma membranes and disorganize the cytoskeleton protein as actin filaments

The Cell Membrane is the Main Target of Resveratrol as Shown by Interdisciplinary Biomolecular/Cellular and Biophysical Approaches

One of the research lines developed in our laboratory is focused on the study of the bioactivity of natural substances. Resveratrol (RV) is a polyphenol nonflavonoid compound present in a number of plant species but mainly in the berries of the red grape Vitis vinifera. The powerful antioxidant action of this molecule is well documented. In this work we evaluated the effects of this substance by adopting diverse experimental strategies. In particular, we studied the effects on cell vitality and cycle by MTT and cytofluorimetric assays. In addition, we explored the action of RV on the cell membrane by a well-consolidated biophysical approach: electrorotation. This technique allows assessment of the structure/function of the cell membrane. The results presented here demonstrate that RV shows a modest effect on the biological properties of the cell in terms of cytotoxicity and cell cycle alterations. On the contrary, a significant effect on the membrane structure/function was observed, consisting of an enhanced intramembrane ion transport. The implications and interpretation of these membrane alterations are discussed