Michela Mazzoni

Synthesis of new indole-based bisphosphonates and evaluation of their chelating ability in PE/CA-PJ15 cells.

Bisphosphonates are the most important class of antiresorptive agents used against osteoclast-mediated bone loss, and, more recently, in oncology. These compounds have high affinity for calcium ions (Ca(2+)) and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. They are extensively used in healthcare, however they are affected by severe side effects; pharmacological properties of bisphosphonates depend on their molecular structure, which is frequently the cause of poor intestinal adsorption and low distribution. In this work we synthesized six novel bisphosphonate compounds having a variably substituted indole moiety to evaluate their extra- and intracellular calcium chelating ability in PE/CA-PJ15 cells. Preliminary in silico and in vitro ADME studies were also performed and the results suggested that the indole moiety plays an important role in cell permeability and metabolism properties.

Blood Lipids in Antarctic and in Temperate-Water Fish Species

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 ω-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, ω-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.

The cytosolic calcium concentration is affected by S‐nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+]c in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+]c, although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S‐nitrosocysteine (CysNO) at low (16 μM) and at high (160 μM) concentrations by measuring the [Ca2+]c by the Fura 2‐AM method. Thapsigargin (TG), which inhibits sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+]c, whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+]c in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+]c homeostasis by stimulating the movement of the ion from the cytosol to other compartments.

Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells

A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

Effect of FSH and progesterone on human spermatozoa cytosolic calcium

Ejaculated spermatozoa must undergo a number of modifications before fertilizing the oocyte: among these the capacitation and the acrosome reaction. Calcium signals play an essential role in these functional and structural modifications. Mature spermatozoa have few organelles and a very small cytoplasmic volume but maintain the homeostasis of [Ca(2+)](c) with great accuracy. We study Ca(2+) mobilization in human spermatozoa exposed to FSH and progesterone by measuring the [Ca(2+)](c) with the FURA-2AM method and report for the first time that the exposure to FSH (up to 98ng/ml) produced an increase of [Ca(2+)](c) to an extent comparable to that observed with 1muM progesterone. FSH and progesterone increase the spermatozoa [Ca(2+)](c) by acting primarily on calcium entry from the external medium. The effects of the two hormones on [Ca(2+)](c) were similar but not identical; the pre-treatment of progesterone blocks the effects of FSH, but not vice-versa. The increase of [Ca(2+)](c) due to FSH was more sensitive to nifedipine (VOCCs inhibitor) than that of progesterone. The effects of these hormones on calcium homeostasis may be relevant for sperm activation.

Olive Oil Phenols and Nitric Oxide Affect Lymphomonocyte Cytosolic Calcium

Publisher Summary The nutritional value of any foods is not limited to their content in essential nutrients or in nutrients generally speaking. Of course, food is a source of energy and essential nutrients, but it is also a mixture of many molecules, some in high, low, or very low amounts. These may interact in various ways and may influence human health in yet unpredictable manners either in the short or long term. For this reason, efforts are being made towards the understanding of the physiological and medical significance of food. The chief constituent of alimentary fat is triacylglycerol. Various triacylglycerol molecules may differ in the proportion and position of the fatty acids they contain. Much work has been done on this subject, especially with reference to polyunsaturated essential fatty acid. However, olive oil contains a number of molecules besides triacylglycerols. This chapter is dedicated to some of these, namely to phenols. Biochemical action of phenols is important for several reasons. Technically speaking, oils can be prepared to contain higher or lower amounts of phenols; moreover, these substances could be used to enrich food or as supplements for the prevention of important illnesses, such as coronary heart disease. The mechanism(s) responsible for the effects of phenols have not yet been fully clarified. Antioxidant activity and free radical scavenging may be relevant in this connection. Yet, other hypotheses have been put forward. For instance, it has been reported that red wine phenols increase cytosolic calcium ([Ca2+]c) in bovine endothelial cells, similarly to bradikinin and to ATP. This chapter reports the effect of extra virgin olive oil phenols on cytosolic calcium [Ca2+]c variations due to nitric oxide (NO).