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Objectives Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signalling pathways comprising cancer inhibition and tumorigenesis. The present study aims to evaluate SIRT2 and SIRT3 gene expression and potential redox reactions in patients with multiple myeloma (MM) at onset and its correlation with disease status, extent and presence of organ damage secondary to myeloma. Design & Methods Total RNA was extracted from 17 MM patients and 10 controls to assess gene expression using real-time PCR. The NAD+/NADH ratio as well as the levels of glutathione peroxidase (GPx) and hydrogen peroxide (HP) in peripheral blood mononuclear cells (PBMCs) were determined using established biochemical assays. Results SIRT2 and SIRT3 expression is reduced in MM patients compared to healthy controls. Correlational analysis demonstrated that SIRT2 reduction is associated with advanced clinical stage and with more advanced bone lesions than in the remaining patients. SIRT3 expression is correlated with lytic bone lesions. Biochemical analysis indicated an imbalance of oxidative stress biomarkers with low concentrations of the antioxidant enzyme GPx, low amounts of NAD + and higher concentrations of pro-oxidant enzyme HP in PBMCs of MM patients compared to controls. Moreover, MM patients with bone lesions had lower concentrations of NAD + and GPx in PBMCs than patients without signs of bone disease. In addition, MM patients had higher quantities of intracellular HP than controls. Conclusions Our results demonstrate that SIRT2 and SIRT3 are downregulated in MM and that lower concentrations correlate with an advanced stage of disease and redox imbalance. We conclude that SIRT2 and SIRT3 together with oxidative stress biomarkers, may be useful for improved risk stratification of MM patients.

TBI is the main cause of death and disability in individuals aged 1–45 in Western countries. One of the main challenges of TBI at present is the lack of specific diagnostic biomarkers, especially for mild TBI (mTBI), which remains currently difficult to value in clinical practice. In this context MiRNAs may be important mediators of the profound molecular and cellular changes that occur after TBI in both the short and the long term. Recently, plasma miRNAs profiling in human TBI, have revealed dynamic temporal regulation of miRNA expression within the cortex. Aim of this study was to select a specific miRNAs panel for mTBI, by focusing the research on the prognostic meaning of miRNAs in the hours following the trauma, in order to be able to use this MIRNAs as potential biomarkers useful for monitoring the follow up of mild TBI. Serum levels of 17 miRNAs were measured by RT-quantitative polymerase chain reaction (qPCR) in 20 patients with mTBI at three different time-points (0h, 24h, 48h) and in 10 controls. For 15 miRNAs we found a significant differences in the comparison among the three time points: for each of these miRNAs the values were greater at baseline and progressively reduced at 24h and 48h. These data allow us to consider the miRNAs included in panel as sensitive and specific biomarkers for mTBI, useful in monitoring the post-trauma period.

Sirtuins are a family of 7 histone deacetylases largely involved in the regulation of cell proliferation, survival and death. The role of sirtuins in tumorigenesis and cancer progression has been previously studied in certain cancer types. Few studies have investigated sirtuin expression in gliomas, with controversial results. The aim of the present study was to investigate the expression of sirtuin-1 (Sirt-1) in diffuse astrocytoma [low grade astrocytoma (LGA)], anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) and in primary glioma cell lines: PLGAC (primary LGA cells); PAAC (primary AA cells); and PGBMC (primary GBM cells). Tumor samples were obtained from patients who underwent craniotomy for microsurgical tumor resection at the Neurosurgery Unit of the University of Messina between 2011 and 2014. Sirt-1 expression was qualitatively analyzed in 30 human glial tumor samples and 5 non-neoplastic brain tissue (NBT) specimens using immunohistochemistry and western blotting techniques. Sirt-1 expression was quantitatively analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Sirt-1 expression in primary cell lines was investigated by immunoblotting and RT-qPCR. Sirt-1 expression was downregulated in gliomas compared to NBTs. Sirt-1 levels also varied among different tumor grades, with more evident downregulation in high-grade (P

Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13-24% range, C18:1n9 in the 20-52% range, and C18:2n6 in the 10-25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct diagnosis.

Hippo signaling pathway is considered a key regulator of tissue homeostasis, cell proliferation, apoptosis and it is involved in cancer development. In skeletal muscle, YAP, a downstream target of the Hippo pathway, is an important player in myoblast proliferation, atrophy/hypertrophy regulation, and in mechano-trasduction, transferring mechanical signals into transcriptional responses. We studied components of Hippo pathway in muscle specimens from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy type 2A and type 2B and healthy subjects. Only DMD muscles had decreased YAP1 protein expression, increased LATS1/2 kinase activity, low Survivin mRNA expression and high miR-21 expression. In light of our novel results, a schematic model is postulated: low levels of YOD1 caused by increased inhibition by miR-21 lead to an increase of LATS1/2 activity which in turn augments phosphorylation of YAP. Reduced amount of active YAP, which is also a target of increased miR-21, causes decreased nuclear expression of YAP-mediated target genes. Since it is known that YAP has beneficial roles in promoting tissue repair and regeneration after injury so that its activation may be therapeutically useful, our results suggest that some components of Hippo pathway could become novel therapeutic targets for DMD treatment.

Inducible nitric oxide synthase knock-out (iNOS(-/-)) mice are valid models of investigation for the role of iNOS in patho-physiological conditions. There are no available data concerning neuroactive amino acid levels of iNOS(-/-) mice and their behaviour in response to pentylenetetrazole (PTZ). We found no significant differences in the convulsive dose 50 (CD(50)) between iNOS(-/-) and control (iNOS(+/+)) mice, however, iNOS(-/-) mice reach the kindled status more slowly than control, suggesting that in basal condition the GABA-benzodiazepine inhibitory inputs are unaltered by iNOS mutation. Clear differences between iNOS(+/+) and iNOS(-/-) mice amino acid concentrations were evident both in basal conditions and after kindling. Our results show that aspartate was significantly lower in all brain areas studied except the brain stem whereas glutamate and glutamine were significantly higher in the cortex, hippocampus and brain stem. GABA was slightly and not significantly higher in the cortex, hippocampus and brain stem, whereas taurine was significantly higher in all areas except diencephalon and glycine was significantly lower in the diencephalon and cerebellum. In this context, the inability of iNOS(-/-) mice to increase the NO levels following PTZ administrations indicate that NO might play a pro-epileptogenic role in the genesis and development of some types of epilepsy. Since there is no correlation between neurotransmitter levels and the development of kindling, it is possible to exclude that the difference between the two strains is due to an imbalance between the considered neurotransmitters, and it is then possible that this difference is due to the presence of iNOS, which might be involved in long term plasticity of the brain.

Interleukin-6-deficient (IL-6 −/− ) mice and their normal littermate (WT) were studied to evaluate their susceptibility to seizures induced by electroshock and audiogenic stimuli at different ages. No significant changes in maximal electroshock susceptibility were evidenced between the two strains, while audiogenic seizures (AGS) can be induced only in IL-6 −/− mice. The effects of age and genetic condition on AGSs were evaluated. The behavioural and electrocortical changes during audiogenic stimulus were observed. In addition, the levels of neurotransmitter amino acids in five brain areas (of both strains) were measured at 60 days of age. Aspartate level significantly increased in the brain stem (BS) and hippocampus (HI), while it decreased in the diencephalon (DE) of IL-6 −/− mice. Glutamate content significantly decreased in the cerebellum (CB), DE and HI. GABA levels significantly decreased in all the areas studied. Glycine significantly decreased in the BS, CB and DE, while taurine decreased only in the DE. The levels of glutamine significantly decreased in all the areas examined, except in the cortex (CX). The changes of neuroactive amino acid levels, particularly in the BS, might explain the characteristic of high propensity to AGS of IL-6 −/− mice. The present data support the validity of IL-6 −/− mice as a novel epileptic model for the study of the pathophysiology and pharmacology of epilepsy.

Telomere shortening is thought to contribute to premature senescence of satellite cells in Duchenne muscular dystrophy (DMD) muscle. Telomeric repeat binding factor-1 (TRF1) and poly (ADP-ribose) polymerase-1 (PARP1) are proteins known to modulate telomerase reverse transcriptase (TERT) activity, which controls telomere elongation. Here we show that an age-dependent telomere shortening occurs in DMD muscles and is associated to overexpression of mRNA and protein levels of TRF1 and PARP1. TERT expression and activity are detectable in normal control muscles and they slightly increase in DMD. This is the first demonstration of TRF1 and PARP1 overexpression in DMD muscles. They can be directly involved in replicative senescence of satellite cells and/or in the pathogenetic cascade through a cross-talk with oxidative stress and inflammatory response. Modulation of these events by TRF1 or PARP1 inhibition might represent a novel strategy for treatment of DMD and other muscular dystrophies.

Genetic animal models have contributed significantly to our understanding of epilepsy causes. Lethargic mice are considered a valid model of absence epilepsy, which have been shown to possess behavioral, electrographic and pharmacological profiles similar to those of humans with absence epilepsies. Single gene mutations that comprise the β 4 subunit of voltage-sensitive Ca 2+ channels underlie the spontaneous discharges of the absence, non-convulsive seizures of lethargic mice. There are no available data concerning how the mutant channels actually behave at terminals in response to chemical activation by subconvulsant stimulation with pentylenetetrazole. In this study, we found no significant difference in the convulsive dose 50 between lethargic and control mice. Lethargic mice showed a more rapid development of kindling to pentylenetetrazole than control animals. No significant differences were observed between the groups of mice rechallenged with pentylenetetrazole 30 or 60 days after the end of the chronic treatment. Marked differences in brain amino acid levels were found between the two strains of mice in basal conditions and after kindling. In conclusion, our results indicate that lethargic mice show a range of biochemical and behavioral changes, correlated in particular with a higher susceptibility to develop kindled seizures.

To elucidate tetraiodothyronine (T4) metabolism in developing rat retina 5-monodeiodinating and 5′-monodeiodinating activities were studied. T4 was incubated with aliquots of homogenate or crude primary subcellular fraction, and the 3,3′,5′-triiodothyronine (rT3) or 3,5,3′-triiodothyronine (T3) produced were measured by radioimmunoassay. Reaction rates were dependent on incubation time, tissue amount, temperature and pH. The optimum pH values were 7.8 and 7.2 respectively for rT3 forming and T3-forming systems. Conversion of T4 to either T3 or rT3 was dependent on dithiothreitol concentration, and the T4-5′-deiodioatrag activity was inhibited by propylthiouracil. Deiodinase activities were mainly found in the crude microsomes. The retinal 5′-monodeiodination rate of T4 was immeasurably low by the 2nd day and the highest values were reached on 15th day of postnatal development. On the other hand deiodination of the T4 tyrosyl ring shows a progressive decline from birth, and adult values were reached on the 15th day. Data support the hypothesis that, in developing rat thyroxine, phenolic and tyrosyl-ring deiodinase activities are present in the retina and their reciprocal changes may regulate morphological and biochemical cell maturation.

Sulpiride, a benzamide derivative neuroleptic, was shown to significantly increase glutamic acid decarboxylase activity in substantia nigra and corpus striatum in either acutely or chronically injected male rats. Hypophysectomy completely prevented this effect suggesting an involvement of an anterior pituitary factor in the central action of sulpiride. Prolactin might possibly mediate the effects of sulpiride since it is known to increase prolactin secretion by an action at the level of the anterior pituitary. Consistent with this hypothesis was the finding of a similar increase in nigral and striatal glutamic acid decarboxylase activity in hyperprolactinemic animals in which an anterior pituitary had been implanted under the kidney capsule.

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.

The effects of acute and chronic injections of haloperidol, sulpiride and apomorphine on serum prolactin (PRL) levels and medial basal hypothalamus (MBH) glutamic acid decarboxylase (GAD) activity were investigated in male rats. Parallel changes in PRL and GAD activity were observed in acutely treated animals. Conversely, a return to normal of the GAD activity associated with high plasma PRL levels was induced by chronic haloperidol and sulpiride treatment. Results are discussed in the light of the possible existence of a hypothetical PRL-gamma-aminobutyric acid (GABA) subsidiary feedback loop.

S-Adenosylmethionine decarboxylase from rat retina is similar to that isolated from other rat tissues with regard to kinetic parameters. pH optimum, putrescine requirement, and sensitivity to spermine. The enzymic activity increases during the first 7 days of postnatal life but decreases until the 20th day. After this period AdoMet decarboxylase activity increases, to reach the highest values at the 90th day. This behavior suggests that such enzymic activity is responsible for spermidine and spermine levels in rat retina and that a high content of retinal spermine might have a role in the photoreceptor outer segment renewal.

The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and gamma-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9-12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S-Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.

Cysteine oxidase activity has been determined in developing rat retina. Enzymic activity is present in 55, 000 × g supernate and in the crude mitochondria. Activity is rather low at birth; but increases with age; in mitochondria it reaches its maximum value at the 25th day while in supernate it increases more rapidly, reaching its maximum value 20 days after birth; unlike in the mitochondria, the activity of supernate considerably decreases during further development. The reason and significance of the postnatal changes in the mitochondrial cysteine oxidase activity are briefly discussed in relation with taurine formation.

Hypotaurine oxidase activity was determined in the primary and secondary subfractions of ox retina. About 78% of enzyme activity was found in the soluble fraction while about 22% was associated with particulate components. In the secondary subcellular fractions about 55% of enzyme activity, recovered from crude mitochondria, was present in the synaptosomal fraction. This finding suggests that the conversion of hypotaurine to taurine is possible in ox retina.

Repeated but not single injections of estradiol benzoate significantly reduced nigral glutamic acid decarboxylase (GAD, EC 4.1.1.15). A single injection of the catecholestrogen 2-hydroxyestradiol produced similar results. Tolerance developed to the latter effect, as reflected by the lack of nigral GAD activity changes in rats repeatedly injected with 2-hydroxyestradiol. Repeated injection of the antiestrogen tamoxifen not only failed to antagonize the action of estradiol benzoate but itself reduced nigral GAD activity. Hypophysectomy, which itself decreased nigral GAD activity prevented the lowering effects of either repeated estradiol benzoate administration or single 2-hydroxyestradiol injection on the enzymatic activity.

The effects of i.v.c. injection of human and salmon calcitonin on biochemical and behavioral parameters related to the extrapyramidal motor system, were investigated in male rats. Calcitonin injection resulted in a potentiation of haloperidol-induced catalepsy and a partial prevention of apomorphine-induced hyperactivity. Moreover calcitonin induced a significant decrease in nigral GAD activity but no change in striatal DA and DOPAC concentration or GAD activity. The results are discussed in view of a primary action of calcitonin on the striatonigral GABAergic pathway mediating the DA-related behavioral messages of striatal origin.

Taurine is present in high concentrations in the retina (1–7) where it constitutes 40–50% of the total aminoacid pool (4, 6).

Methionine adenosyltransferase (MAT) activity was evaluated in cerebral cortex and cerebellum in controls and in rats treated with thyroxine. In controls the enzyme showed a different pattern in cerebral cortex and cerebellum during neonatal and late suckling periods. Hyperthyroid rats showed a significant increase of the enzyme in cerebral cortex only at the 2nd day of the neonatal period; in cerebellum the developmental pattern of MAT in neonatal period was anticipated temporally by 2–4 days. During the late suckling period thyroxine treatment produced in cerebellum a significant decrease in MAT activity at the 15th day after birth. From these data, we propose that hyperthyroidism may cause precocious induction of MAT both in cerebral cortex and in cerebellum and that the increased availability of S-adenosyll-methionine during the neonatal period could be related to its utilization also in polyamine biosynthesis.