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Merkel cell carcinoma (MCC) is an aggressive skin cancer, with a propensity for early metastasis. Therefore, early diagnosis and the identification of novel targets become fundamental. The enzyme nicotinamide N-methyltransferase (NNMT) catalyzes the reaction of N-methylation of nicotinamide and other analogous compounds. Although NNMT overexpression was reported in many malignancies, the significance of its dysregulation in cancer cell phenotype was partly clarified. Several works demonstrated that NNMT promotes cancer cell proliferation, migration, and chemoresistance. In this study, we investigated the possible involvement of this enzyme in MCC. Preliminary immunohistochemical analyses were performed to evaluate NNMT expression in MCC tissue specimens. To explore the enzyme function in tumor cell metabolism, MCC cell lines have been transfected with plasmids encoding for short hairpin RNAs (shRNAs) targeting NNMT mRNA. Preliminary immunohistochemical analyses showed elevated NNMT expression in MCC tissue specimens. The effect of enzyme downregulation on cell proliferation, migration, and chemosensitivity was then evaluated through MTT, trypan blue, and wound healing assays. Data obtained clearly demonstrated that NNMT knockdown is associated with a decrease of cell proliferation, viability, and migration, as well as with enhanced sensitivity to treatment with chemotherapeutic drugs. Taken together, these results suggest that NNMT could represent an interesting MCC biomarker and a promising target for targeted anti-cancer therapy., (© 2024. The Author(s).)

An adequate placental vascularization allows the proper development of the fetus and it is crucial for the gestational success. A number of factors regulate angiogenesis, including vascular endothelial growth factor (VEGF), which induces the synthesis of nitric oxide (NO), a potent vasodilator produced by three different nitric oxide synthase (NOS) isoforms. NO is essential to maintain a low vascular resistance in the fetoplacental circulation, although at high concentrations, it may combine with excess superoxide to produce peroxynitrite, which reacts with proteins giving rise to nitrotyrosine. Since obesity, whose incidence is increasing worldwide, is characterized by a low-grade inflammatory state and increased levels of oxidative and nitrative stress, both affecting placental function, our aim was to evaluate the expression of VEGF, eNOS, and iNOS in full-term placentas obtained from normal weight and pre-pregnancy obese women by means of immunohistochemistry and real-time PCR. Moreover, we assessed the NO levels and the nitrotyrosine immunoexpression in the same sample groups. Our results show a significantly higher immunohistochemical expression of VEGF and eNOS in the endothelium of placentas from obese women than in controls, whereas the immunoexpression of iNOS was comparable in the two groups. These data agree with those of the gene expression analysis, thus suggesting the possible existence of a compensatory mechanism for changes in placental blood flow associated with obesity. As concerns nitrotyrosine and NO levels, we observed a significant increase in placental tissue from obese women which may contribute to the development of metabolic and cardiovascular diseases both in the mother and the offspring.

Impaired male fertility may have a variety of causes, among which asthenozoospermia. In its etiology, several bioactive substances, such as cytokines may be involved. In this context, our aim was to evaluate the expression of interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor-1α, in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients. We evaluated twenty-eight infertile patients affected by idiopathic asthenozoospermia and twenty-three normospermic fertile donors, age-matched. Sperm parameters were evaluated; immunohistochemical analysis and enzyme-linked immunosorbent assay were then performed in isolated spermatozoa. Spermatozoa from the asthenozoospermic group presented an increased expression of IL-1β, COX-2, and HIF-1α compared with the normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence sperm motility. Nevertheless, further studies are needed in order to assess whether these bioactive mediators have a potential relevance as targets in future therapeutic strategies for the treatment of male infertility.

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca(2+) concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.

In order to investigate the molecular mechanisms of the inhibition of Na+/K+-ATPase in Gestational Hypertension (GH), we incubated Na+/K+-ATPase purified from human placenta of 6 healthy normotensive women with plasma from 6 GH women and 6 healthy controls. We determined the enzyme activity by the method of Esman, and the anthroyl-ouabain-binding capacity, dissociation constant (Kd) and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl-ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5- hexatriene (TMA-DPH). The addition of total and protein-free GH plasma to normal Na+/K+-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1:100), as well as the ouabain-binding capacity, Kd and tau. GH plasma significantly decreased the fluorescence polarization and lifetime values of TMA-DPH. These observations indicate that the inhibition caused by GH plasma on Na+/K+-ATPase might be due to a reduction of the number of active molecules or a modification of the ouabain-binding site suggesting the existence of digitalis-like factor. A link between the modification of the lipid moiety of the enzyme and the Na+/K+-ATPase inhibition might be hypothesized.

A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.

It has been recently hypothesized that in PIH a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidation products into the circulation, with subsequent damage of endothelial cell membranes. In this hypothesis the endothelial cell and further increase in circulating lipoperoxide levels, which are by themselves able to induce smooth muscle constriction and increased pressor responsiveness to angiotensin II. In order to investigate this issue, we studied the basal content of lipid peroxides in terms of malondialdehyde (MDA) in the syncytiotrophoblast plasma membranes (SPM) from PIH women. Moreover, we investigated the susceptibility to peroxidation of SPM using an in vitro oxidative stress as a tool to verify the predisposition to the in vivo development of peroxidation products. The fatty acid composition of the membranes was also analyzed. Microvillus membrane lipoperoxide concentrations were significantly increased in PIH women (62.8 +/- 7.6 ng MDA/mg prot) compared with healthy pregnant subjects (37.6 +/- 4.8 ng MDA/mg prot; p < 0.01). The formation of TBARS under the action of phenylhydrazine was significantly greater in PIH women (90.3 +/- 7.4 mmol MDA/mol cholesterol) than in normal pregnant subjects (68.6 +/- 6.4 mmol MDA/mol cholesterol; p < 0.01). In PIH microvillus membrane we also observed a significant increase of the content of polyunsaturated arachidonic acid. The increased susceptibility to oxidative stress of SPMs from PIH women might be due either to reduced antioxidant systems or to an abnormality of the lipid composition of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)