The possible role of the hydroxyl group of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, and crystallographic studies. Three representative cosubstrates have been used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, the enzyme follows a rapid equilibrium random bi−bi mechanism with a rate-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe yields a 14-fold decrease of kcat, while it does not change appreciably the affinity of the H site for the substrate. In this case, it would appear that the role of the hydroxyl function is to stabilize the transition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bonding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M. W. (1997) Biochemistry 36, 576−585]. Moreover, no structural differences are observed between the Y108F mutant and the wild type, suggesting that the removal of the hydroxyl group is solely responsible for the loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of Tyr 108 yields an approximately 7-fold increase of kcat and a constant kcat/ value. Lack of a critical hydrogen bond between 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appears to be the basis of the increased kcat. In the 1-chloro-2,4-dinitrobenzene/GSH system, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glutathione transferase P1-1 catalysis, depending on the nature of the electrophilic cosubstrate.
The direct role of antiphospholipid antibodies (aPL) at maternal-fetal interface has not been fully investigated, especially whether they are involved in physiological and pathological implantation conditions, in an antiphospholipid syndrome (APS)-independent manner. In fact, trophoblast cells and placental endothelial cells at the implantation site express potential aPL targeted-phospholipid antigens (PL Ags); thus, the local production and presence of their specific antibodies, not related to APS (characterized by aPL presence in the peripheral blood), could be a potential marker of aberrant invasion, implantation and fetal-maternal immune tolerance processes.Anti-Beta2glycoprotein I (anti-β2GPI) and anticardiolipin (aCL Ab) antibodies (the most clinically relevant aPL) were detected by immunoenzymatic assay (ELISA), in the amniotic fluid (AF) of 167 women with physiological and complicated common pregnancy conditions, sharing an aberrant implantation process, such as recurrent pregnancy loss (RPL), autoimmune hypothyroidism (ahT) and smoking. All women included in the study were negative to peripheral blood aPL.aCL and anti-β2GPI antibodies were detectable in all the AF samples. RPL, ahT and smoking patients had higher level of anti-β2GPI Abs (IgM) compared to women with physiological pregnancies (p < 0.0001). Since IgM cannot cross the placenta, their local production in response to maternal-fetal interface stimuli, could be hypothesized.The presence of aPL in the AF (not related to APS) could reveal a potential clinical significance at maternal-fetal interface in selected pregnancy complications, in which an aberrant implantation process, and in turn an impaired fetal-maternal immune tolerance cross-talk, could occur.
Olfactory dysfunction is a prevalent condition associated with post-viral syndromes, chronic rhinosinusitis, and neurodegenerative diseases. While olfactory cleft sampling has been investigated in neurology, its potential for assessing localized biomarkers in rhinological conditions remains underexplored. This study presents a minimally invasive nasal brushing technique specifically targeting the olfactory cleft, enabling simultaneous biomarker quantification and cytological analysis. A comprehensive literature review was performed to highlight its potential utility in both clinical practice and research.
Self-preservation is a typical property of living organisms, observed in the simplest prokaryotic cell as well as in the more complex pluricellular organisms. Surprisingly we found a self-preservation mechanism operating at the level of a single enzyme. Human glutathione transferase P1-1 operates in such a way towards either killer compounds (competitive and irreversible inhibitors) or physical factors (temperature and UV-rays), which could suppress its detoxicating and anti-cancer activity in the cell. This property, here termed 'co-operative self-preservation', is based on a structural intersubunit communication, by which one subunit, as a consequence of an inactivating modification, triggers a defence arrangement in the other subunit. Paradoxically this ability, developed during evolution for the survival of the cell, may not always be advantageous for us. In fact, glutathione transferase P1-1 is overexpressed in most tumour cells and pharmacological attempts to inhibit this enzyme in vivo, to prevent the drug resistance phenomenon during chemotherapy, may be thwarted by such self-preservation.
Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide. Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease. MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146. We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression. Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression. Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series. For some patients, we also performed the time courses of molecular monitoring. Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment. We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular “warning” marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.
Lactoferrin (Lf), a multifunctional cationic glycoprotein synthesized by exocrine glands and neutrophils, possesses an in vitro antiviral activity against SARS-CoV-2. Thus, we conducted an in vivo preliminary study to investigate the antiviral effect of oral and intranasal liposomal bovine Lf (bLf) in asymptomatic and mild-to-moderate COVID-19 patients. From April 2020 to June 2020, a total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided into three groups. Thirty-two patients (14 hospitalized and 18 in home-based isolation) received only oral and intranasal liposomal bLf; 32 hospitalized patients were treated only with standard of care (SOC) treatment; and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, untreated, healthy subjects were added for ancillary analysis. Liposomal bLf-treated COVID-19 patients obtained an earlier and significant (p < 0.0001) SARS-CoV-2 RNA negative conversion compared to the SOC-treated and untreated COVID-19 patients (14.25 vs. 27.13 vs. 32.61 days, respectively). Liposomal bLf-treated COVID-19 patients showed fast clinical symptoms recovery compared to the SOC-treated COVID-19 patients. In bLf-treated patients, a significant decrease in serum ferritin, IL-6, and D-dimers levels was observed. No adverse events were reported. These observations led us to speculate a potential role of bLf in the management of mild-to-moderate and asymptomatic COVID-19 patients.
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