Michela Codini

Nitric oxide metabolites, prostaglandins and trigeminal vasoactive peptides in internal jugular vein blood during spontaneous migraine attacks

Despite evidence emerging from the experimental model of nitroglycerin-induced headache, the endogenous increase in nitric oxide (NO) production during migraine attacks is only speculative. It has been hypothesized that there is a close relationship between activation of the l-arginine/NO pathway and production of certain vasoactive and algogenic prostaglandins during spontaneous migraine attacks, but this suggestion also needs to be confirmed. In the present study the levels of nitrites, the stable metabolites of NO, were determined with high performance liquid chromatography (HPLC) in the internal jugular venous blood of five patients affected by migraine without aura examined ictally. These samples were taken within 30 min, 1, 2, and 4 h from the onset of the attack and at the end of the ictal period. At the same time, the plasma levels of calcitonin gene-related peptide (CGRP), neurokinin A (NKA), prostaglandin E2 (PGE2) and 6 keto PGF1α, the stable product of PGI2, were assessed with radioimmunoassay (RIA) kits in the same samples. The levels of the intracellular messengers, cGMP and cAMP, were also measured with the RIA method. Nitrite, cGMP, CGRP and NKA levels reached their highest values at the first hour, then they tended to decrease progressively and returned, after the end of attacks, to values similar or below those detected at the time of catheter insertion (anova, statistical significance: P < 0.001; P < 0.002; P < 0.002; P < 0.003, respectively). PGE2 and 6 keto PGF1α, as well as cAMP levels also significantly increased at the first hour but reached a peak at the 2nd hour and remained in the same range until the 4th and 6th hours. Then their values tended to decrease after the end of attacks, becoming lower than those measured immediately after catheter positioning for internal jugular venous blood drawing (anova: P < 0.002, P < 0.004, P < 0.001, respectively). Our results support early activation of the l-arginine/NO pathway which accompanies the release of vasoactive peptides from trigeminal endings and a late rise in the synthesis of prostanoids with algogenic and vasoactive properties which may intervene in maintaining the headache phase.

Radioligand binding assay of M1–M5 muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes

Analysis of lymphocyte muscarinic cholinergic receptors using quantitative techniques such as radioligand binding assay is made difficult due to the low density of these sites and the lack of subtype-specific selectivity of most available muscarinic ligands. In this study, a combined kinetic and equilibrium labeling technique recently developed for brain tissue was used for labeling the five muscarinic cholinergic receptor subtypes in intact human peripheral blood lymphocytes. No specific muscarinic M1 receptor binding was detectable in human peripheral blood lymphocytes using [3H]-pirenzepine as a ligand. Labeling of M2-M5 muscarinic receptors using [3H]N-methyl-scopolamine (NMS) by occluding various receptor subtypes with muscarinic antagonist and mamba venom resulted in the labeling of M2-M5 receptors in brain as well as in human peripheral blood lymphocytes. The relative density of different receptor subtypes was M3 > M5 > M4 > M2. The development of a reproducible technique for assaying muscarinic cholinergic receptor subtypes expressed by human peripheral blood lymphocytes may contribute to clarify their role in lymphocyte function.

First pilot newborn screening for four lysosomal storage diseases in an Italian region: Identification and analysis of a putative causative mutation in the GBA gene

We report the first newborn screening pilot study in an Italian region for four lysosomal disorders including Pompe disease, Gaucher disease, Fabry disease and mucopolysaccharidosis type 1. The screening has been performed using enzymatic assay on Dry Blood Spot on filter paper. A total of 3403 newborns were screened. One newborn showed a reduction of β-glucosidase activity in leucocytes. Molecular analysis revealed a status of compound heterozygous for the panethnic mutation N370S and for the sequence variation E388K, not yet correlated to Gaucher disease onset. The functional consequences of the E388K replacement on β-glucosidase activity were evaluated by in vitro expression, showing that the mutant protein retained 48% of wild type activity. Structural modeling predicted that the E388K replacement, localized to a surface of the enzyme, would change the local charges distribution which, in the native protein, displays an overwhelming presence of negative charges. However, the newborn, and a 4 year old sister showing the same genomic alterations, are currently asymptomatic. This pilot newborn screening for lysosomal diseases appears to be feasible and affordable to be extended to large populations. Moreover other lysosomal diseases for which a therapy is available or will be available, could be included in the screening.

Antioxidative capacity of Lactobacillus fermentum LF31 evaluated in vitro by oxygen radical absorbance capacity assay.

OBJECTIVE The aim of this study was to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells. METHODS The antioxidant capability of L. fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the oxygen radical absorbance capacity method. RESULTS The analysis revealed that the interaction of probiotic strain cells supplemented with a prebiotic exerts a remarkable antioxidant capacity. CONCLUSION The L. fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.

VERY LONG CHAIN FATTY ACID SPHINGOMYELIN IN NUCLEAR LIPID MICRODOMAINS OF HEPATOCYTES AND HEPATOMA CELLS: CAN THE EXCHANGE FROM C24:0 TO C16:0 AFFECT SIGNAL PROTEINS AND VITAMIN D RECEPTOR?

The 24:0 sphingomyelin of nuclear lipid microdomains from normal cells shifts to 16:0 sphingomyelin in nuclear lipid microdomains from cancer cells. The narrower microdomains in the nucleus are associated with the changes to proteins involved in hepatocarcinogenesis.

Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinsons disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinsons disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinsons disease and other neurodegenerative disorders.

Gentamicin Arrests Cancer Cell Growth: The Intriguing Involvement of Nuclear Sphingomyelin Metabolism

The use of gentamicin for the treatment of bacterial infection has always been an interesting and highly speculated issue for the scientific community. Conversely, its effect on cancer cells has been very little investigated. We studied the effect of high doses of gentamicin on non-Hodgkin’s T-cell human lymphoblastic lymphoma (SUP-T1). We showed that gentamicin delayed cell growth and induced cell death in lymphoma cells with a rather mild effect on lymphocytes. In SUP-T1 cells, GAPDH, B2M, CDKN1A and CDKN1B were down-expressed in comparison with lymphocytes. Gentamicin treatment in SUP-T1 cells restored the expression of GAPDH, B2M and CDKN1A to values similar to those of lymphocytes and caused overexpression of CDKN1B. The drug acted via sphingomyelin metabolism; in whole cells, sphingomyelinase activity was stimulated, whereas in purified nuclei, sphingomyelinase activity was inhibited and that of sphingomyelin-synthase was stimulated, with a consequent high level of nuclear sphingomyelin content. We suggest that the increase of nuclear sphingomyelin might enrich the nucleus of lipid microdomains that act as a platform for active chromatin and, thus, might be responsible for gene expression. It is possible that in lymphoblastic lymphoma, high doses of gentamicin induce a beneficial therapeutic outcome.

Nuclear Lipid Microdomain as Resting Place of Dexamethasone to Impair Cell Proliferation

The action of dexamethasone is initiated by, and strictly dependent upon, the interaction of the drug with its receptor followed by its translocation into the nucleus where modulates gene expression. Where the drug localizes at the intranuclear level is not yet known. We aimed to study the localization of the drug in nuclear lipid microdomains rich in sphingomyelin content that anchor active chromatin and act as platform for transcription modulation. The study was performed in non-Hodgkin’s T cell human lymphoblastic lymphoma (SUP-T1 cell line). We found that when dexamethasone enters into the nucleus it localizes in nuclear lipid microdomains where influences sphingomyelin metabolism. This is followed after 24 h by a cell cycle block accompanied by the up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase inhibitor 1B (CDKN1B), growth arrest and DNA-damage 45A (GADD45A), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes and by the reduction of signal transducer and activator of transcription 3 (STAT3) and phospho signal transducer and activator of transcription 3 (phoshoSTAT3) proteins. After 48 h some cells show morphological changes characteristic of apoptosis while the number of the cells that undergo cell division and express B-cell lymphoma-2 (Bcl-2) is very low. We suggest that the integrity of nuclear lipid microdomains is important for the response to glucocorticoids of cancer cells.

High-performance liquid chromatographic method for the determination of prolyl peptides in urine

A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.

Impact of Gravity on Thyroid Cells

Physical and mental health requires a correct functioning of the thyroid gland, which controls cardiovascular, musculoskeletal, nervous, and immune systems, and affects behavior and cognitive functions. Microgravity, as occurs during space missions, induces morphological and functional changes within the thyroid gland. Here, we review relevant experiments exposing cell cultures (normal and cancer thyroid cells) to simulated and real microgravity, as well as wild-type and transgenic mice to hypergravity and spaceflight conditions. Well-known mechanisms of damage are presented and new ones, such as changes of gene expression for extracellular matrix and cytoskeleton proteins, thyrocyte phenotype, sensitivity of thyrocytes to thyrotropin due to thyrotropin receptor modification, parafollicular cells and calcitonin production, sphingomyelin metabolism, and the expression and movement of cancer molecules from thyrocytes to colloids are highlighted. The identification of new mechanisms of thyroid injury is essential for the development of countermeasures, both on the ground and in space, against thyroid cancer. We also address the question whether normal and cancer cells show a different sensitivity concerning changes of environmental conditions.