Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

Pneumonectomy with and without induction chemo-radiotherapy for non-small cell lung cancer: short and long-term results from a single centre.

BACKGROUND AND OBJECTIVES:   Pneumonectomy for non small cell lung cancer (NSCLC) after induction radio-chemotherapy (IT) has been associated with high peri-operative risk and its safety and efficacy is still debated. The aim of this retrospective study was to compare short and long-term results of pneumonectomy in patients treated with and without IT (radiotherapy plus chemotherapy) for NSCLC. MATERIALS AND METHODS: From 1995 to 2008, 85 consecutive patients underwent pneumonectomy: 49 received pre-operative radiotherapy and chemotherapy (IT group), and 36 patients did not (non-IT group). Peri-operative and long-term outcomes were compared. RESULTS: Major complications rate was 14.3% for IT group and 16.7% for non-IT group (p = n.s.). Mortality rate was 2% in IT group and 5.5% in non-IT group (p = n.s.). Post-operative hospital stay was significantly longer in the IT group (p < 0.0001) as the need for blood transfusion (p = 0.002). Indeed, the mortality rate was similar in the left- and right-sided operations. 5 years survival was 45.3% for IT group and 38.4% for non-IT group (p = n.s.) and 5 year disease free survival rates were 42.3% vs. 37.8% for the two groups, respectively (p = n.s.). Among the clinical, surgical and pathological features no differences on long term outcomes were found with regards to IT. DISCUSSION: Pneumonectomy is a feasible and safe procedure even after pre-operative IT. Our results showed a prolonged hospitalization and the need for blood transfusion in the IT group.

Trocar vs. Seldinger small bore pleural drains: does the technique influence the outcomes? A prospective single-centre study.

OBJECTIVE: The aim of this study is to compare two positioning techniques of 12-French (Fr) thoracic drains in terms of efficacy, safety, and patient comfort. PATIENTS AND METHODS: This is a prospective, non-randomized, competitive, non-inferiority study comparing the Seldinger vs. Trocar technique. The primary endpoint was an analysis of the factors that led to unsuccessful drainage positioning. Between the two groups, clinical variables, procedure times, pain, and complications were compared. RESULTS: Seventy-two patients were enrolled in group 1 (Seldinger) and 45 in group 2 (Trocar). The mean procedural time was 7.93±3.02 min vs. 7.09±3.67 min, respectively (p: 0.33). The mean VAS for procedural pain was 2.22±1.47 vs. 2.80±1.88, p: 0.07, and the mean at day 2 was 3.6±1.2 in the SBWGD group vs. 2.7±1.1 in the Unico Group (p: 0.04). There was no difference in terms of complications, residual effusion, and pneumothorax at the first post-procedural chest X-ray. Four days after the procedure, the drain removal rate was 11.6% in group 1 vs. 25% in group 2 p: 0.063). The chest tube was removed after a mean period of 8.87±7.20 days after resolution of pleural effusion or tube dislodgement (7 cases in group 1 vs. 11 in group 2, p: 0.053). CONCLUSIONS: The two techniques resulted in comparable pain and complication rates. Both drains are well-tolerated and efficient at draining pleural effusion, with very low rates of complications and failure. We recommend inserting a longer tube for patients who require chest drainage for an extended period of time.

Pulmonary malacoplakia at early stage: use of polymerase chain reaction for detection of Rhodococcus equi.

Malacoplakia is a rare inflammatory condition characterized by the accumulation of benign macrophages associated with pathognomonic Michaelis-Gutmann bodies (MGBs). It is usually found in the genito-urinary tract, and has been associated with immunocompromised states. In this short report, we present 5 patients with pulmonary nodules clinically suspicious for primary or metastatic lung cancer. The histologic examination of the surgical specimens revealed a nonspecific granulomatous chronic disease, and despite the paucity of classical MGBs, a pulmonary malacoplakia was suspected. In all cases the opportunistic pathogen Rhodococcus equi (R. equi) was identified by 16S rRNA gene sequence analysis, leading to the final pathological diagnosis of malacoplakia. We conclude that pulmonary malacoplakia associated with R. equi is a rare disease affecting also immunocompetent patients. The pathogenesis and the diagnostic problems are discussed. Since infection by R. equi is treatable, the importance of its early recognition should be emphasized.

Malignant solitary fibrous tumour of the chest wall: a challenging case.

Solitary fibrous tumors are very rare neoplasms that seldomly appear in extra-serosal soft tissues. In such cases, an accurate preoperative diagnosis is often difficult and challenging, especially in extrapleural ones. Traditionally, extrapleural solitary fibrous tumours have been regarded as indolent neoplasms similar to their intra-thoracic counterparts, although there has been some evidence that this subgroup could be a subset of more aggressive malignant tumours. For these reasons, surgical excision is mandatory and represents, to date, the best therapeutic option. In this article we report a case of a malignant solitary fibrous tumor of the chest wall in a 58-year-old man. Problems related to differential diagnosis and the possible pitfalls that can be encountered in the diagnostic process of such rare tumors are discussed.

[Clinical effect of bovine pericardial strips on air leak after stapled pulmonary resection in "frail" patients: early results]. / Impiego clinico di suturatrici rivestite con pericadio bovino nella prevenzione delle perdite aeree postoperatorie in pazienti "fragili" sottoposti a resezione polmonare.

AIM: Postoperative air leaks and in particular persistent air leaks (>5 days) after pulmonary resection still represent a common complication and the first cause of hospital stay delay. Aim of this experimental trial was to investigate the efficacy of the use of bovine pericardium strips (in terms of reduction of postoperative leakage and hospital stay) in "critical" patients (COPD, emphysema etc.) who underwent pulmonary resection. METHODS: From October 2010 to February 2011, eight patients (experimental group, Group A) were preoperative selected and underwent pulmonary resection with bovine pericardium strips (Peri-Strips Dry; Synovis ). The inclusion criteria of a "frail patient" were established by a dedicate pneumologist according with clinical and functional data (predicted postoperative FEV1 ranging from 35% and 80% of the theorical predicted value). For comparison, from January 2010 to September 2010, we retrospectively reviewed the data of 28 patients who satisfied the same inclusion criteria and underwent pulmonary resection with standard surgical procedures. This group of patients represents our control group (Group B). RESULTS: There were no significant differences between the two groups in age, gender, preoperative risk factors for developing a postoperative air leak, preop FEV1 and type of resection. No technical deficiencies in the use of bovine pericardium strips were observed in Group A. Postoperative leakage was significant different in the two groups being persistent air leak detected in 0% in Group A versus 17.8% of Group B (P=0.046). Consequently, chest tube duration (6.75±0.84 days [Group A] vs. 9.70±1.26 days (Group B), P=0.019) and hospital stay (10.13±0.83 days [Group A] vs. 12.95±1.37 days [Group B], P=0.013) were lower in the experimental group. CONCLUSION: Bovine pericardium strips are safe and easy-to-do technique to reduce postoperative air leaks after pulmonary resection in "critical" patients.

Huge primary pericardial liposarcoma.

Liposarcomas are the second most common soft tissue sarcoma in adults. They occur predominantly in the lower limbs and retroperitoneum, whereas primary mediastinal liposarcomas are extremely rare. Liposarcomas are often asymptomatic and may reach a considerable size before causing any symptoms related to direct invasion or compression of other thoracic organs. We report a case of a 69-year-old woman with a giant primary pericardial liposarcoma causing cardiac tamponade and discuss its clinical and imaging features and surgical treatment and review the literature.

A versatile and compact surface plasmon resonance spectrometer based on single board computer.

The widespread diffusion of low-cost but high-performance hardware is enhancing the realization of scientific equipment with features at the research laboratory level. In this paper, we demonstrate hardware implementation of a surface plasmon resonance compact device with high accuracy and measurement times appropriate for many applications. Image acquisition is realized by a Raspberry Pi single board computer with a camera module, and a Python code is used to process data. A flexible optical setup can work in two different configurations, namely, the inspection mode and angle resolved measurement mode. The inspection mode is used to precisely locate the light-emitting diode interrogation beam on the sample, avoiding uneven or faulty regions. The measurement mode allows us to monitor in real time the position of the minimum reflectivity with subpixel resolution. Performance tests show a resolution in the bulk refractive index of 4.9 × 10-6 refractive index units for 10 s acquisition time.

Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

Cytoskeletal reorganization in skeletal muscle differentiation: from cell morphology to gene expression.

Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.

Clinical features, imaging findings, treatment aspects of elastofibroma dorsi and long-term outcomes after surgical resection.

OBJECTIVE: We aim to present clinical features, imaging findings, treatment aspects of the elastofibroma dorsi (ED), which is a benign tumor arising from connective tissue at the scapular region, and long-term outcomes after surgical resection. PATIENTS AND METHODS: We evaluated retrospectively 82 patients (55 females, 27 males; mean age, 60 years; age range, 23-78 years) with ED who underwent surgery between January 1994 and May 2014; subsequently all patients were invited for follow-up, which consisted of physical and US examinations. RESULTS: Subscapular location was almost constant (79/82 patients). Right, left and bilateral location was noted in 39, 28 and 15 cases, respectively. 52/82 patients were symptomatic. The diagnosis was made on physical examination and imaging studies: 49 ultrasound, 43 computed tomography and 54 magnetic resonance examinations were performed overall. Surgical treatment consisted in marginal excision; in all cases diagnosis was confirmed by histological examination. The mean hospitalization was 3 days, with minor complications. Out of the 82 patients, only 25 gave their consent to follow-up; mean time passed after surgery was 64.7 months; 1 case of local recurrence was suspected by ultrasound and, then, confirmed by magnetic resonance imaging. CONCLUSIONS: In our series, clinical features and imaging findings of ED are consistent with current evidence; however, results of our follow-up group marks a difference from the literature, according to which there is no evidence of local recurrence after complete resection. Diagnosis of ED is based on clinical and imaging features; treatment is surgical, especially in symptomatic cases. Prolonging the clinical and US follow-up period may be useful in identifying local recurrence.

Glutamine transport and enzymatic activities involved in glutaminolysis in human platelets.

Glutamine is actively metabolized in human platelets, representing a preferential mitochondrial oxidative substrate in these cells. The primary importance of this metabolic route of glutamine is further confirmed here by the observation that platelet glutaminase activity is entirely represented by the phosphate dependent glutaminase or glutaminase I, most probably localized in the mitochondrial platelet fraction and classified by kinetic analysis as a kidney-type form. The following step of the glutamine metabolizing pathway, allowing the entrance of the amino acid skeleton carbons in the Krebs cycle, might be catalyzed by both glutamate dehydrogenase and aspartate transaminase, the first being entirely mitochondrial and the latter 65% mitochondrial. We also investigated platelets for the presence of one or more specific transport systems involved in glutamine uptake and we present the first evidence for two glutamine transport systems in human platelets that by inhibition analysis appear to share characteristics with the Na(+)-dependent ASC system and the Na(+)-independent L system for dipolar amino acid uptake. Both systems display affinity characteristics for glutamine in the range of plasma glutamine concentration and may thus have physiological relevance for the uptake of the amino acid in these cells.

pH sensitivity of the thrombin-induced rise in fructose 2,6-bisphosphate content of human platelets.

The stimulation of human platelets with thrombin results in a rapid and sustained increase in the fructose 2,6-bisphosphate content which may play an important role in the potentiation of glycolytic flux induced by the agonist. The investigation of the effect of pH on thrombin-induced rise in platelet fructose 2,6-bisphosphate content is reported here. The results indicate that the early intracellular alkalinization which follows platelet stimulation may contribute to mediate the positive effect of thrombin on the regulatory metabolite.

Bradykinin stimulates fructose 2,6-bisphosphate metabolism in human fibroblasts.

Bradykinin (BK), a peptide released during inflammatory response, has been investigated for its ability to regulate glucose metabolism in human fibroblasts. The peptide is able to significantly increase glycolytic flux in these cells. The strict relationship between the glycolytic rate and the levels of fructose 2,6-bisphosphate (Fru-2,6-P2) strongly suggests that the metabolite plays a key role in the regulation of glucose metabolism by bradykinin. The mechanism by which bradykinin increases Fru-2,6-P2 content involves the activation of 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of the metabolite. The study of the multiple signalling systems triggered by bradykinin demonstrates the involvement of the rise in intracellular Ca2+ concentration and of protein kinase C mediated pathway in the mechanism by which bradykinin increases Fru-2,6-P2 content and PFK-2 activity.

A role for phospholipase D activation in the lipid signalling cascade generated by bradykinin and thrombin in C2C12 myoblasts.

In the present study evidence is provided for a rapid activation of lipid signalling pathways induced by thrombin and bradykinin (BK) in C2C12 myoblasts. Both agonists were able to increase [3H]inositol phosphates (InsP), 1,2-[3H]diacylglycerol (DAG) and [3H]phosphatidic acid (PtdOH) levels. In particular [3H]PtdOH values were rapidly increased and maintained at significantly high values at prolonged times of incubation. BK and thrombin were able to activate phospholipase D (PLD) in vivo as demonstrated by the accumulation of [3H]phosphatidylethanol (PtdEtOH) through the transphoshatidylation reaction catalyzed by the enzyme in the presence of ethanol. The observation that ethanol could significantly reduce [3H]PtdOH formation in myoblasts stimulated with BK and thrombin indicates that stimulation of PLD has a major role. The two agonists appear to stimulate PLD activity through a common molecular mechanism, involving the activation of protein kinase C (PKC). In addition, BK and thrombin appear able to activate DAG kinase at early times of incubation and also this pathway may contribute to determine the increase in [3H]PtdOH levels. This is the first report which describes activation of lipid signalling pathways by BK and thrombin in myoblast cells and it is possible that these early signals may have an important role in mediating the biological effects of the two agonists.

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells.

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.

Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase.

The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.

Dual regulation of sphingosine 1-phosphate-induced phospholipase D activity through RhoA and protein kinase C-alpha in C2C12 myoblasts.

Previous studies showed that in C2C12 cells, phospholipase D (PLD) and its known regulators, RhoA and protein kinase Calpha (PKCalpha), were downstream effectors in sphingosine 1-phosphate (SPP) signalling. Moreover, the role of PKC for SPP-mediated PLD activation and the requirement of PKCalpha for RhoA translocation were reported. The present results demonstrated that inactivation of RhoA, by overexpression of RhoGDP dissociation inhibitor (RhoGDI) as well as treatment with C3 exotoxin, attenuated SPP-stimulated PLD activity, supporting the involvement of RhoA in the stimulation of PLD activity by the bioactive lipid in C2C12 myoblasts. In addition, the effect of PKCalpha inhibitor Gö6976 on the SPP-induced PLD activation in myoblasts, where RhoA function was inactivated, was consistent with a dual regulation of the enzyme through RhoA and PKCalpha. Interestingly, the subcellular distribution of PLD isoforms, RhoA and PKCalpha, in SPP-stimulated cells supported the view that the functional relationship between the two PLD regulators, demonstrated to occur in SPP signalling, represents a novel mechanism of regulation of specifically localized PLD.

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.