Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Increased Toll-like receptor (TLR) mRNA expression in monocytes is a feature of metabolic syndrome in adolescents.

BACKGROUND: Metabolic syndrome (MetSyn) is diagnosed frequently in some but not all overweight adolescents. Chronic inflammation, as seen in obesity, is strongly associated with MetSyn. OBJECTIVES: The aim of this pilot study was to assess the correlation between activation of the innate immune system and MetSyn, independent of body mass index (BMI), in a young population. METHODS: We quantitatively measured both systemic pro-inflammatory cytokines and gene expression of Toll-like receptors (TLRs) and downstream cytokines in circulating monocytes obtained from nine adolescents with metabolic syndrome (Overwt-MetSyn) and eight BMI-matched controls (Overwt-Healthy). RESULTS: The Overwt-MetSyn group demonstrated a significant elevation in expression of TLR2, TLR4, tumour necrosis factor-a (TNF a) and interleukin-6 (IL6) in peripheral monocytes, and increased circulating levels of TNF a and IL6 when compared with the Overwt-Healthy group. TLR2 (r = 0.78, P < 0.001), TLR4 (r = 0.57, P < 0.01) and TNF a (r = 0.61, P < 0.01) gene expression positively correlated with serum levels of TNF a. CONCLUSIONS: Our study suggests that activation of the innate immune pathway via TLRs may be partially responsible for the increased systemic inflammation seen in adolescents with MetSyn.

Vitamin D protects human endothelial cells from H2O2 oxidant injury through the Mek/Erk-Sirt1 axis activation.

Endothelium homeostasis alterations govern the pathogenesis of cardiovascular diseases. Several studies show that vitamins anti-oxidant proprieties rescue the endothelial functions adversely affected by oxidative stress in several diseases. We investigated the vitamin D anti-oxidant potential in human endothelial cells exposed to H2O2 oxidative stress. Vitamin D protected endothelial cells against H2O2 oxidative stress counteracting the superoxide anion generation, the apoptosis and blocking the extrinsic caspase cascade by positively controlling phospho-active ERKs level. MEKs/ERKs inhibitor U0126 reverted the vitamin D anti-oxidant effects. Characterizing the vitamin D downstream effector, we found that vitamin D up-regulated SirT-1 and reverted the SirT-1 down-regulation induced by H2O2. ERKs activation by vitamin D strictly correlated with SirT-1 protein accumulation since both MEKs/ERKs inhibition and ERK1/2 silencing decreased SIRT-1. SirT-1 inhibition by Sirtinol reverted the vitamin D anti-oxidant effects. Thus, vitamin D significantly reduced the endothelial malfunction and damage caused by oxidative stress, through the activation of MEKs/ERKs/SirT-1 axis.

Levels of polychlorinated biphenyls in fish and shellfish from the Adriatic Sea.

Levels of 18 polychlorinated biphenyl (PCB) congeners were determined by gas chromatography-mass spectrometry (GC-MS) in some marine species, living both in the coastal area and in deeper seawater. In some species analysis was performed separately in edible parts (fillets) and in viscera. The existence and degree of bioaccumulation was assessed studying individual species of very different size, with the smaller being younger. Furthermore, with a multivariate statistical analysis, a correlation between PCB congeners and the feeding habits and habitat of the fish was demonstrated. The results show that fat from edible parts (fish fillets) had total PCB levels in the range 22.6-601.9 µg kg⁻¹ (with 601.9 µg kg⁻¹ in anchovies), while fat from viscera showed much higher concentrations (407.3-916.6 µg kg⁻¹). Bioaccumulation was confirmed, comparing PCB levels between younger and older individual hake, squid, and horned octopus. The total PCB concentration ratio (older/younger individuals) ranges from 2.11 (squid = 292.1/137.8 µg kg⁻¹) to 3.46 (hake = 546.0/158.0 µg kg⁻¹).

Total antioxidant capacity (TAC): is it an effective method to evaluate the oxidative stress in uraemia?

Lipoprotein abnormalities are common in uraemia and are considered important factors for development of atherosclerosis and progression of renal disease. Reduction of total antioxidant capacity (TAC) and lipid peroxidation (LP) probably play a major role in both processes. The aim of this study was to assess the effect of renal function, dietary manipulation and lipids on TAC of uraemic patients with different chronic renal failure (CRF). Sixty patients (36M, 24F), aged 60 +/- 12 years were divided into five groups according to serum creatinine levels (sCr,mg/dl)--CRFI, 1.5-3; CRFII, > 3-5.5; CRFIII, > 5.5; CRFIV, > 3 on vegetarian supplemented diet (SD); CRFV haemodialysis patients (HD)- and investigated for TAC by enhanced chemiluminescent assay, autoantibodies against oxidized LDL (oxLDLAb), lipids, apolipoprotein AI, B, Lp(a) and uric acid (UA). The results were compared to a control group of 19 people (8M, 11F), aged 52 +/- 11 years with sCr < 1.5. TAC increased significantly with the progression of CRF and was strongly related to both sCr and UA. Lipids and SD did not show any influence on TAC. Unexpectedly, lipid peroxidation did not correlate to TAC, neither to sCr or UA. HD accounted for a mild reduction of both TAC and LP. Patients on SD showed a marked reduction of LP as compared to patients with a similar degree of renal failure (CRF-III) but on conventional diet. Our results suggest that elevated TAC in uraemia is likely to be dependent on increased UA levels and does not seem to induce an effective protection in vivo from oxidative stress. In conclusion, TAC does not appear to be a reliable method for assessing the oxidative susceptibility of CRF patients.

Nutritional and prognostic correlates of bioimpedance indexes in hemodialysis patients.

We carried out a cross sectional and longitudinal study to assess whether bioimpedance indexes (resistance, Rz; reactance, Xc; phase angle, PA) reflect the nutritional status of hemodialysis (HD) patients, and bear a significant association with their long-term survival. The bioimpedance data of 131 patients on chronic HD treatment were compared with those of 272 healthy controls matched for age and sex. Nutritional status was assessed by anthropometric variables, serum albumin (SA), normalized protein catabolic rate (nPCR), and subjective global assessment (SGA). All three bioimpedance indexes varied significantly with HD treatment, however, with the exception of Xc in post-HD, they were on average significantly (P < 0.016) different from controls either pre- and post-HD. Post-HD PA appeared to be the best index of nutritional status, being significantly correlated with SA, age, mid arm muscle circumference (MAMC), SGA, and nPCR (R2 = 0.44; P < 0.01). However, depending on the cut-off levels, PA failed to detect clinically overt malnutrition in one to two thirds of the patients with the worst SGA score. During the follow-up the changes in bioimpedance indexes reflected poorly the changes in dry blood weight, only delta Rz bore a significant correlation (r = 0.29; P < 0.01) with delta body wt. Patients having baseline phase angle values within the lower quartile had a significantly lower two-year survival rate than patients having higher values (59.3% vs. 91.3%; P < 0.01). Cox's analysis (proportional hazard model) showed that phase angle as a predictor of death outweighed all other parameters included in the model (age, SA, nPCR, MAMC, SGA), with a relative risk of 2.6 (95% CI = 1.6 to 4.2). Bioimpedance indexes do not appear to be reliable in detecting clinically overt depletion of lean body mass. However, the strong association of PA with patient survival suggests that this bioimpedance index reflects some dimension of the illness, which is not fully identifiable with the deranged nutritional status.

Enhanced expression of myogenic regulatory genes in aging skeletal muscle.

MyoD, myogenin, myf-5, and MRF4, belonging to the family of basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs), control muscle cell differentiation, in concert with other transcription factors such as MEF-2, yet their role in age-related skeletal muscle alteration has not been addressed. We here report that MyoD and myogenin transcripts are expressed at high levels in the hind limb muscles of newborn mice and their level of expression continuously declines throughout postnatal life to become virtually undetectable in the adult mouse. However, these transcripts are again expressed at high levels in the muscles of older mice. MRF4 transcript, on the other hand, is present at a constant level throughout the life span of the animal. Conversely, the expressions of myf-5 and MEF-2C, components of the autoregulatory loop for the activation of bHLH gene expression, conspicuously increase in adult and senile muscle. In order to establish whether these transcripts are functioning in the aged muscle we investigated the expression of bHLH inhibitory factor Id mRNA showing that it does not present significant changes during aging. Immunofluorescence analysis with an anti-myogenin antibody revealed nuclear accumulation of the protein in the muscle fibers of old, but not of adult, mice. Muscle-specific genes transactivated by MyoD and myogenin such as AChR, MLC, and MCK are also up-regulated during aging, albeit at a lower level. Significant changes in the size and ratio of type I/type II fibers are detectable in senile muscle. These findings show that all members of the MRF family are expressed to a high extent and are likely active in senile muscle. It is conceivable that these changes might operate as a compensatory mechanism in maintaining the expression of differentiated muscle products in senile muscle at a steady-state level.

Differential modulation of expression of the two acylphosphatase isoenzymes by thyroid hormone.

The modulation of expression of the skeletal muscle and erythrocyte acylphosphatase isoenzymes by thyroid hormone has been investigated. Our results indicate a differential regulation of the two enzymic isoforms by tri-iodothyronine (T3) in K562 cells in culture: an increase in the specific mRNA during T3-stimulation is shown only for the skeletal muscle isoenzyme. A fast and transient T3 induction of the accumulation of the specific mRNA can be observed, reaching a maximum 8 h after hormone treatment and then rapidly decreasing almost to the steady-state level after 24 h. A nuclear run-on assay was performed to explore the mechanisms of this regulation. These studies indicate that T3 induction of skeletal muscle acylphosphatase mRNA is due, at least in part, to a fast and transient increase in the rate of gene transcription, within 4 h after hormone administration. A very rapid decrease is then observed within a further 2 h. T3-dependent accumulation of the mRNA for the skeletal muscle acylphosphatase requires ongoing protein synthesis, as confirmed by inhibition with cycloheximide or puromycin. These findings indicate that the transcriptional regulation of the gene may be indirect.

The success of closed reduction in the treatment of complex developmental dislocation of the hip.

The purpose of our study was to determine the efficacy of closed reduction in the treatment of complex developmental dislocation of the hip. We identified two factors, the cone of stability and the limbus type, through the use of arthrography and gentle examination under anesthesia, which are useful guidelines in the management of complex developmental dislocation of the hip. We feel as a result of this study we can select those cases of complex developmental dislocation of the hip that are amenable to closed reduction and separate them from those other cases for which we recommend open reduction.

Expression of growth arrest-specific (gas) genes in senescent murine cells.

The growth arrest-specific (gas) genes were initially identified on the basis of their preferential expression in mouse fibroblasts during quiescence, followed by down-regulation upon reentry into the cell cycle. We here report studies on the expression of these genes in murine fibroblasts undergoing replicative senescence in vitro. Our results indicate a different behavior between senescent and GO-arrested quiescent fibroblasts. Expression of the gas1 and 6 genes was dramatically reduced in senescent cells. Only basal levels of gas2, 3, and 5 genes were detected in senescent fibroblasts, and they were independent of the growing conditions of the cultures. Down-regulation of the gas1 gene expression in senescent cells was apparently due to reduced transcription of the gas1 gene. This correlates with an altered pattern of factors that bind to the promoter region of the gas1 gene, as measured by band shift assay with nuclear extracts of senescent fibroblasts.

A general-purpose system for long-term recording from a microelectrode array coupled to excitable cells.

A PC-based system for acquisition and processing of data from excitable cells on a microelectrode array is described. Simple and low-cost amplification and filtering custom stages are used. A software package for processing acquired data is proposed.

Early detection of cell metabolism with a silicon microsensor.

A silicon microsensor (ISFET--Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.

Early detection of cell metabolism with a silicon microsensor.

A silicon microsensor (ISFET - Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.

Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development.

The growth arrest-specific gas5 gene was isolated from mouse genomic DNA and structurally characterized. The transcriptional unit is divided into 12 exons that span around 7 kb. An alternative splicing mechanism gives rise to two mature mRNAs which contain either 11 or 12 exons, and both are found in the cytoplasm of growth-arrested cells. In vivo, the gas5 gene is ubiquitously expressed in mouse tissues during development and adult life. In Friend leukemia and NIH 3T3 cells, the levels of gas5 gene mRNA were high in saturation density-arrested cells and almost undetectable in actively growing cells. Run-on experiments indicated that the gas5 gene is transcribed at the same level in both growing and arrested cells. On the other hand, in dimethyl sulfoxide-induced differentiating cells a sharp decrease in the rate of transcription was observed shortly before the cells reached the postmitotic stage. These results indicate that in density-arrested cells accumulation of gas5 mRNA is controlled at the posttranscriptional level while in differentiating cells expression is regulated transcriptionally.

Anti-hepatitis C virus epidemiological study in two dialysis centers in Florence.

We studied the prevalence and incidence of anti-hepatitis C virus (HCV) antibodies in 350 patients during 15 months and looked for some risk factors. We found a significant correlation between anti-HCV positivity and length of dialysis treatment and treatment in more than one center. We propose some prophylactic rules.

Regulation of expression of growth arrest-specific genes in mouse fibroblasts.

The suppression of growth arrest-specific (gas) gene expression by serum appeared to be independent of protein synthesis, but expression in resting cells was sensitive to 2-aminopurine, an inhibitor of intracellular protein kinases. Although accumulation of gas gene mRNA was reduced by serum, nuclear transcription of the gas-2, -3, and -5 genes was observed in serum-stimulated cells, indicating that posttranscriptional events may regulate mRNA levels. Growth induction by serum, on the other hand, led to suppression of transcription of the gas-1 gene. Cell cycle regulation and the serum response of gas-1 were lost in ras-transformed cells.

Spontaneous channel activity induced by tumor promoter TPA in chick myotubes.

Patch-clamp recordings were used to study the activation of ion channels in the cell membrane of cultured embryonic chick myotubes treated with the specific activator of protein kinase C, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 1 x 10(-7) M). Myotubes exhibited a spontaneous channel activity when the TPA-induced dedifferentiative processes developed. This consisted in the activation of inward current channels (approximately 35 pS conductance; approximately 6 ms open time). These spontaneously active channels were insensitive to alpha-bungarotoxin, curare, atropine and tetrodotoxin and were not inhibited by the withdrawal of TPA. It is suggested that a prolonged stimulation of the protein kinase C causes a irreversible deregulation of the membrane channel function during cell dedifferentiation.

Traumatic dislocation and fracture dislocation of the hip. A long-term follow-up study.

Of 84 fracture dislocations of the hip reviewed, 73 were posterior fracture dislocations, nine were central, and two were obturator. Fifty-five were followed from one to 30 years (average, 4 1/2 years). The patients' ages ranged from three to 83 years (average, 33.1 years). All had primary attempt at closed reduction, with 15 treated with subsequent open reduction (six immediate and nine delayed). Satisfactory results were obtained in 42% of hips treated with closed reduction and 40% of those treated with open reduction. Statistical analysis of the results was done. A stable closed reduction produced results as satisfactory as open reduction. When followed for a sufficiently long time interval, severe fracture dislocations, regardless of treatment mode, caused unsatisfactory results.

pH in the Jelly Layer of Starfish Oocytes: (starfish oocytes/jelly layer/pH).

Using liquid ion exchanger micro-electrodes we have studied the pH in the vicinity of starfish oocytes and eggs and also in their jelly layers. Although highly variable, the pH in these zones was consistently lower (6.3-7.6) than the pH of sea water (7.8-8.2). Aged oocytes/eggs presented more acid environments than fresh oocytes. The pH indicator Phenol Red shows that the ovarian fluid is more acid than seawater, ranging from 6.0 to 7.5. Our data suggests that starfish oocytes, from their time in the ovary until several hr after their release, are surrounded by an acid environment. This micro-environment may play a role in metabolic repression in the ovary and in the regulation of sperm-egg interaction following spawning.

Biofiltration in elderly uremic patients.

We treated five elderly patients with conventional hemodialysis (CH) or biofiltration (BF) to establish whether their dialytic tolerance was better. For three patients treatment time was reduced from twelve to nine hours a week; for the other two, treatment time remained unchanged (10.5-12 h/week) because of their high interdialytic weight gain. At the beginning and end of the study, clinical status, biochemical data, nutritional status and acid-base balance (ABB) were checked. Plasma levels of small molecules, potassium and phosphate were unchanged for all patients. All had a lower number of episodes or less severe hypotension and good control of ABB. No patients had metabolic alkalosis or worsened nutritional status. For all patients BF was an efficacious choice compared to CH, giving them good health.