A polymorphism upstream MIR1279 gene is associated with pericarditis development in Systemic Lupus Erythematosus and contributes to definition of a genetic risk profile for this complication.

Recently, a study has shown that a polymorphism in the region of MIR1279 modulates the expression of the TRAF3IP2 gene. Since polymorphisms in the TRAF3IP2 gene have been described in association with systemic lupus erithematosus (SLE) susceptibility and with the development of pericarditis, our aim is to verify if the MIR1279 gene variability could also be involved. The rs1463335 SNP, located upstream MIR1279 gene, was analyzed by allelic discrimination assay in 315 Italian SLE patients and 201 healthy controls. Moreover, the MIR1279 gene was full sequenced in 50 patients. A case/control association study and a genotype/phenotype correlation analysis were performed. We also constructed a pericarditis genetic risk profile for patients with SLE. The full sequencing of the MIR1279 gene in patients with SLE did not reveal any novel or known variation. The variant allele of the rs1463335 SNP was significantly associated with susceptibility to pericarditis ( P = 0.017 and OR = 1.67). A risk profile model for pericarditis considering the risk alleles of MIR1279 and three other genes (STAT4, PTPN2 and TRAF3IP2) showed that patients with 4 or 5 risk alleles have a higher risk of developing pericarditis ( OR = 4.09 with P = 0.001 and OR = 6.04 with P = 0.04 respectively). In conclusion, we describe for the first time the contribution of a MIR1279 SNP in pericarditis development in patients with SLE and a genetic risk profile model that could be useful to identify patients more susceptible to developing pericarditis in SLE. This approach could help to improve the prediction and the management of this complication.

Polymorphisms in STAT-4, IL-10, PSORS1C1, PTPN2 and MIR146A genes are associated differently with prognostic factors in Italian patients affected by rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease resulting in chronic inflammation of the synovium and consequent cartilage and bone erosion. RA is associated strongly with the presence of rheumatoid factor (RF), and consists of clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and -negative patients. This study was designed to evaluate whether relevant single nucleotide polymorphisms (SNPs) associated with RA and other autoimmune disorders are related to RF, ACPA and clinical phenotype in a cohort of biologic drugs naive Italian RA patients; 192 RA patients and 278 age-matched healthy controls were included. Clinical and laboratory data were registered. We analysed a total of 12 single nucleotide polymorphisms (SNPs) in signal transducer and activator of transcription-4 (STAT-4), interleukin (IL)-10, psoriasis susceptibility 1 candidate 1 (PSORS1C1), protein tyrosine phosphatase, non-receptor type 2 (PTPN2), endoplasmic reticulum aminopeptidase 1 (ERAP1), tumour necrosis factor receptor-associated 3 interacting protein 2 (TRAF3IP2) and microRNA 146a (MIR146A) genes by allelic discrimination assays. Case-control association studies and genotype/phenotype correlation analyses were performed. A higher risk to develop RA was observed for rs7574865 in the STAT-4 gene, while the rs1800872 in the IL-10 gene showed a protective effect. The presence of RF was associated significantly with rs1800872 variant in IL-10, while rs2910164 in MIR146A was protective. ACPA were associated significantly with rs7574865 in STAT-4. The SNP rs2233945 in the PSORS1C1 gene was protective regarding the presence of bone erosions, while rs2542151 in PTPN2 gene was associated with joint damage. Our results confirm that polymorphisms in STAT-4 and IL-10 genes confer susceptibility to RA. For the first time, we described that SNPs in PSORS1C1, PTPN2 and MIR146A genes were associated differently with a severe disease phenotype in terms of autoantibody status and radiographic damage in an Italian RA population.

Miglustat Reverts the Impairment of Synaptic Plasticity in a Mouse Model of NPC Disease.

Niemann-Pick type C disease is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol within the late endolysosomal compartment of cells and accumulation of gangliosides and other sphingolipids. Progressive neurological deterioration and insurgence of symptoms like ataxia, seizure, and cognitive decline until severe dementia are pathognomonic features of the disease. Here, we studied synaptic plasticity phenomena and evaluated ERKs activation in the hippocampus of BALB/c NPC1-/- mice, a well described animal model of the disease. Our results demonstrated an impairment of both induction and maintenance of long term synaptic potentiation in NPC1-/- mouse slices, associated with the lack of ERKs phosphorylation. We then investigated the effects of Miglustat, a recent approved drug for the treatment of NPCD. We found that in vivo Miglustat administration in NPC1-/- mice was able to rescue synaptic plasticity deficits, to restore ERKs activation and to counteract hyperexcitability. Overall, these data indicate that Miglustat may be effective for treating the neurological deficits associated with NPCD, such as seizures and dementia.

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

Phospholipase-like myotoxins induce rapid membrane leakage of non-hydrolyzable ether-lipid liposomes.

Two phospholipase-like myotoxins--ammodytin L from Vipera ammodytes and myotoxin II from Bothrops asper--are shown to be able to induce leakage of liposomes made from non-hydrolyzable ether-linked phospholipids. This demonstrates that the cytolytic activity of these toxins is completely independent of any remaining enzyme activity or contamination with active phospholipases.

Effect of ammodytin L from Vipera ammodytes on L-6 cells from rat skeletal muscle.

Ammodytin L (AMDL) is a myotoxic phospholipase-like protein from the venom of Vipera ammodytes with a serine in position 49 instead of an aspartate, therefore this toxin is devoid of phospholipase activity, and the membrane-damaging effect does not involve any step of phospholipase activity. The aim of the present study was to analyze the effect of AMDL on L-6 cells from rat skeletal muscle to investigate its mechanism of action and the role of calcium ions in its muscle-damaging activity. Our data indicate that the effect of ammodytin L is strongly dependent on the degree of cell differentiation. Low doses of myotoxin gave rise to a marked release of creatine kinase in myotubes differentiated from L-6 myoblasts and the presence of calcium ions plays a role in the cytotoxic effect. The presence of EGTA in the incubation buffer reduced by 50% the release of creatine kinase. No membrane damage was observed in myoblasts, but there was a significant increase of intracellular calcium concentration measured with Fura-2. A non-specific membrane effect of AMDL was ruled out using platelets as reference cells: no platelet aggregation pattern and no increase in intracellular calcium were observed.

Evidence of domain formation in cardiolipin-glycerophospholipid mixed monolayers. A thermodynamic and AFM study.

The interaction of the three main components of the mitochondrial membrane, namely cardiolipin, phosphatidylcholine, and phosphatidylethanolamine, has been studied investigating mixed cardiolipin-phosphatidylcholine and cardiolipin-phosphatidylethanolamine monolayers at different cardiolipin molar fractions. The thermodynamic behavior of the mixed monolayers was investigated by means of surface pressure and surface potential measurements, and atomic force microscopy was employed to characterize the morphology of the monolayers. Langmuir isotherms and surface potential curves show a regular behavior with a progressive transition toward the isotherm of the pure component. Positive deviations from ideality in the excess Gibbs energies of mixing suggest the presence of repulsive interactions in both systems. Analysis of partial molecular dipole moment indicates a discontinuity at a definite cardiolipin/phosphatidylethanolamine molar fraction, suggesting the formation of a stoichiometric complex; as a consequence, in mixed cardiolipin-phosphatidylethanolamine monolayers, a phase separation is observed at phosphatidylethanolamine excess. AFM measurements indicate the presence of two domains: one made by phosphatidylethanolamine and the other by a regular arrangement of phosphatidylethanolamine and cardiolipin at a fixed molecular ratio.

Adenosine- and 2-chloro-adenosine-induced cytopathic effects on myoblastic cells and myotubes: involvement of different intracellular mechanisms.

We recently suggested that, in muscular dystrophies, the excessive accumulation of adenosine as a result of an altered purine metabolism may contribute to progressive functional deterioration and muscle cell death. To verify this hypothesis, we have taken advantage of C2C12 myoblastic cells, which can be differentiated in vitro into multinucleated cells (myotubes). Exposure of both proliferating myoblasts and differentiated myotubes to adenosine or its metabolically-stable analog, 2-chloro-adenosine, resulted in apoptotic cell death and myotube disruption. Cytotoxicity by either nucleoside did not depend upon extracellular adenosine receptors, but, at least in part, by entry into cells via the membrane nitro-benzyl-thio-inosine-sensitive transporter. The adenosine kinase inhibitor, 5-iodotubercidin, prevented 2-chloro-adenosine-induced (but not adenosine-induced) effects, suggesting that an intracellular phosphorylation/activation reaction plays a key role in 2-chloro-adenosine-mediated cytotoxicity. Conversely, adenosine cytotoxicity was aggravated by the addition of homocysteine, suggesting that adenosine effects may be due to the accumulation of S-adenosyl-homocysteine, which blocks intracellular methylation-dependent reactions. Both nucleosides markedly disrupted the myotube structure via an effect on the actin cytoskeleton; however, also for myotubes, there were marked differences in the morphological alterations induced by these two nucleosides. These results show that adenosine and 2-chloro-adenosine induce apoptosis of myogenic cells via completely different metabolic pathways, and are consistent with the hypothesis that adenosine accumulation in dystrophic muscles may represent a novel pathogenetic pathway in muscle diseases.

Abnormal urinary excretion of glycosaminoglycans in Albers-Schönberg disease.

Abnormal urinary excretion of acid mucopolysaccharides in two children affected with Albers-Schönberg disease is described. The urine specimens were first checked for their acid glycosaminoglycan (AGAG) content by a screening test. The AGAGs were then evaluated by uronic acid determination using the carbazole method, and the ratio between high molecular mass and low molecular mass AGAGs was determined. Qualitative characterization, before and after enzymatic digestion with chondroitinases AC (ChAC) and ABC (ChABC), of the total AGAGs contained in the specimen and of each AGAG separated with Dowex 1X2 was also performed by electrophoresis. The pattern of the urinary mucopolysaccharides in the two patients showed an abnormal quantity of heparan sulphate (HS) and a smaller quantity of dermatan sulphate (DS). Such abnormal urinary AGAGs excretion should confirm the assumption of some authors that malignant infantile osteopetrosis may be caused by a deficiency in the lysosomal enzymatic activity, or by some anomaly in the cell membrane function.

A new screening test for the diagnosis of mucopolysaccharidoses.

A simple method for the electrophoretic separation of glycosaminoglycans from untreated urine is presented. It is shown that this rapid electrophoresis on specially shaped acetate cellulose strips gives more information than other screening tests used in laboratory for discriminating between normal and pathological samples. This technique is useful particularly with low levels of glycosaminoglycans which may give false-negative responses.

Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.

A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8-32 microM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.

Zinc status in athletes: relation to diet and exercise.

Zinc is involved in the biochemical processes supporting life, such as cellular respiration, DNA reproduction, maintenance of cell membrane integrity and free radical scavenging. Zinc is required for the activity of more than 300 enzymes, covering all 6 classes of enzyme activity. Zinc binding sites in proteins are often of distorted tetrahedral or trigonal bipyramidal geometry, made up of the sulphur of cysteine, the nitrogen of histidine or the oxygen of aspartate and glutamate, or a combination. Zinc in proteins can either participate directly in chemical catalysis or be important for maintaining protein structure and stability. The nutritional habits of elite athletes during training and competition are quite different from the recommended diet in the majority of the population. Endurance athletes often adopt an unusual diet in an attempt to enhance performance: an excessive increase in carbohydrates and low intake of proteins and fat may lead to suboptimal zinc intake in 90% of athletes. Mild zinc deficiency is difficult to detect because of the lack of definitive indicators of zinc status. In athletes, zinc deficiency can lead to anorexia, significant loss in bodyweight, latent fatigue with decreased endurance and a risk of osteoporosis.

Hyperhomocyst(e)inemia is associated with carotid atherosclerosis.

The atherogenicity of homocyst(e)ine--H(e) --emerged from many studies showing an association between moderately elevated levels and vascular occlusive disease. The aim of this study was to evaluate whether high homocyst(e)ine levels were associated with carotid atherosclerosis. Carotid atherosclerosis was defined as an intimal media thickness of internal and carotid bifurcation of at least 2 mm on the near and far walls as determined by B-mode ultrasonography. The study population included 91 patients: group 1 (61% males, mean age 64+/-10 years, 57% with history of hypertension) with ultrasound evidence of carotid atherosclerosis and 100 with normal carotid walls--group 2 (36% males, mean age 52+/-15 years, 27% with history of hypertension). Homocyst(e)ine levels (mol/L) were determined by high-performance liquid chromatography with a fluorescent detector. Body mass index, dyslipidemia, smoking, diabetes, serum creatinine, plasma folic acid and vitamin B12 were not significantly different in the two groups. Homocyst(e)ine levels (micromol/L) were significantly higher in patients with carotid ather osclerosis than in those with normal arteries (11.7+/-6.5 micromol/L, 95% CI 10.4-13.1 vs 8.07+/-4.4 micromol/L, 95% CI 7.2-8.9, p<0.0001). By multiple regression analysis H(e) levels were positively correlated with male gender (p<0.02), age (p<0.001), and negatively with folic acid (p<0.0001). By logistic regression the independent predictors of carotid atherosclerosis were male gender (OR 2.65), hypertension (OR 2.55), age (x10 years, OR 2.15) and H(e) levels (x1 micromol/L, OR 1.11). This study confirmed homocyst(e)ine is associated with carotid atherosclerosis. Consequently the authors recommend H(e) levels be screened in all patients at risk for atherosclerosis.

Physical exercise intensity can be related to plasma glutathione levels.

The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity.

Physical exercise intensity can be related to plasma glutathione levels.

The aim of the present study was to examine the effect of different kinds of physical exercise on plasma glutathione levels. Male Wistar rats were randomly divided into four groups: In walking group (W; n=6), rats were trained to walk 0.8 m/min for 45 min; slow running group (SR; n=6) were trained to run 4 m/min for 45 min; fast running group (FR; n=6) ran 8 m/min for 60 min and control rats (C; n=6) remained in their home cages. All animals were sacrificed after exercise and the levels of reduced glutathione (GSH) in plasma samples determined by high performance liquid chromatography (HPLC) with a fluorescent detector. Compared to controls, exercise did not change GSH plasma levels of the W group. A tendency to decrease blood GSH was observed in plasma samples of the SR group and in the FR group, physical exercise resulted in a dramatic decrease in GSH plasma levels. These data suggest that during light physical exercise there is a low production of reactive oxygen species (ROS) with a low request for antioxidant defence such as oxidation of GSH. The dramatic decrease observed in GSH levels in FR rats would indicate the presence of oxidative stress able to modify blood antioxidant profiles. Our results suggest that GSH plays a central antioxidant role in blood during intensive physical exercise and that its modifications are closely related to exercise intensity.

[Non-ketotic hypoglycemia caused by carnitine palmitoyl transferase 1 deficiency]. / Ipoglicemia non chetotica da deficit di carnitina palmitoil trasferasi 1.

A 9-year-old girl was referred to our hospital after recurrent episodes of hypoglycemia, altered consciousness and persistent vomiting without acetonemia or myopathic symptoms. Other pertinent laboratory data included elevated BUN, hyperammonemia and very low levels of triglycerides with elevated free fatty acids. The patient was born from unaffected but related parents (second cousins) and the illness was previously diagnosed as Reye encephalopathy. Recurrence of similar attacks suggested an underlying metabolic disorder. Several syndromes of impaired FFA beta oxidation were taken into account and discarded successively after laboratory investigations: systemic carnitine deficiency, Medium and Long Chain Acyl-CoA Dehydrogenase deficiency and Multiple Acyl CoA Dehydrogenation deficiency (Glutaric aciduria, Ethylmalonic-adipic aciduria and riboflavin-responsive multiple acyl CoA dehydrogenation deficiency). Urinary and hematic gas-chromatography and Mass-Spectrometry show no abnormality in Medium Chain fatty acids and in C6-C10 dicarboxylic acids. Carnitine plasma concentrations (both total and free) were above normal levels while in urine acetyl carnitine was low in respect to longer acyclic radicals. Among metabolic defects located at the level of hepatic fatty acid oxidation, only Carnitine Transferase deficiency can explain this peculiar mosaic of data (precursors of the blocked reaction are elevated in blood whereas lack of the metabolites derived uniquely from this reaction explains all the clinical manifestations).

Erythrocyte formimino glutamate transferase in FIGLU aciduria.

A patient is described who presented at an early age with failure to thrive and vomiting, and had a gross excretion of formimino glutamic acid. She had normal concentration of serum folate and vitamin B12, and no haematological abnormalities, and is not mentally retarded. The Michaelis constant for erythrocyte formimino glutamate transferase was in the normal range, but the enzyme behaves differently from that from reference subjects with respect to inhibitors and activators.

[Determination of HbA2 by a microchromatographic method. Reliability and simplification of the use of prepared columns]. / Dosaggio della Hb A2 con metodo microcromatografico. Affidabilità e semplificazione d'uso di colonne preconfezionate.

A modified procedure for hemoglobin A2 determination by microchromatography was developed simplifying an easily commercially available column kit method. Blood samples were analyzed simultaneously by means of DE-52 microchromatography described by Huisman et al. (reference 12) to check the reliability of the new modified method. In the Pediatric Clinic of Cagliari University where the original Huisman method was used the values of HbA2 obtained were as follows: Normal subjects (35 cases) 2.470 +/- 0.351, beta-Thalassemia heterozygous subjects (38 cases) 5.142 +/- 0.342. The modified method gave respectively the following results: Normal subjects 2.578 +/- 0.303, beta-Thalassemia heterozygous subjects 5.465 +/- 0.400. No overlap was found between normal and beta-thalassemia trait samples. It is suggested that the simplified method is very reliable and very simple to organize even in laboratories with few facilities and when needs for only few tests per week exist.

[Nocturnal changes in osteocalcin in children with constitutional growth retardation]. / Variazioni notturne dell'osteocalcina in bambini affetti da ipostaturalità costituzionale.

Growth Hormone (GH) and serum Bone GLA-Protein (BGP or Osteocalcin), a sensitive and specific marker of bone turnover, were measured in 5 children with growth retardation, during 12 h period from 8 p.m. every 30'. A nocturnal periodicity in Osteocalcin was found: BGP rose slightly during sleep in the patients studied, maximum concentration being reached between 3 and 5 a.m. There were no consistent correlations between Osteocalcin concentration and circulating levels of GH in the subjects tested. BGP determination may be of interest in the evaluation of children with short stature but standardized analytical conditions remain to be determined.