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.

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.

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.

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.

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.

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.

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.

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).

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.

Liberation of cyanide from alpha-aminonitriles relative to amygdalin.

A series of aminoitriles have been synthesized and studied whose nonenzymatic dissociation with release of cyanide may be varied by modest alteration of their molecular structure from that obtained with nonenzymatic dissociation of amygdalin to that obtained from enzymatic dissociation of amygdalin by substantial quantities of beta-glucosidase. The relationship between such alterations in molecular structure and nonenzymatic dissociation is discussed. A combination of the results of these studies and studies relating molecular structure to physical localization propensity in tumors has potential in the design of chemotherapeutic agents.

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.

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.

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.

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.

Bone changes in end-stage oxalosis.

Primary hyperoxaluria is a rare metabolic disease characterized by exaggerated production of oxalic acid. Clinically the disease is characterized by recurrent calcium nephrolithiasis, progressive renal failure, and early death in uremia. As the regular dialysis treatment may prolong survival, a new syndrome may develop. This is due to intense and continuous deposition of calcium oxalate crystals in soft and bone tissues. The radiologic aspects of oxalate deposition in four adult patients on chronic renal dialysis with histologic and clinical evidence of massive bone oxalosis are reported.

Effect of chronic propranolol treatment on bone changes of secondary hyperparathyroidism in dialysis patients.

To assess the effect of long-term propranolol therapy on uremic osteodystrophy, we evaluated retrospectively the biochemical, X-ray, and bone histological changes in 9 dialysed hypertensives who had been on propranolol treatment for periods ranging from 1 to 8 years. The control group included 9 well-matched dialysed uremics never treated with beta-blockers. The two groups did not differ in serum ionized calcium or iPTH levels. The incidence of bone resorption evaluated on X-ray findings and on bone biopsy specimens was of a comparable degree in the two groups. Our data show that propranolol even if started early in the course of renal failure, is of no benefit in preventing uremic bone disease.

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.