Neutral lipids as early biomarkers of cellular fate: the case of α-synuclein overexpression.

α-synuclein (α-syn) accumulation and aggregation is a common pathological factor found in synucleinopathies, a group of neurodegenerative disorders that includes Parkinson´s disease (PD). It has been proposed that lipid dyshomeostasis is responsible for the occurrence of PD-related processes, however, the precise role of lipids in the onset and progression of neurodegenerative disorders remains unclear. Our aim was to investigate the effect of α-syn overexpression on neutral lipid metabolism and how this impacts on neuronal fate. We found lipid droplet (LD) accumulation in cells overexpressing α-syn to be associated with a rise in triacylglycerol (TAG) and cholesteryl ester (CE) levels. α-syn overexpression promoted diacylglycerol acyltransferase 2 upregulation and acyl-CoA synthetase activation, triggering TAG buildup, that was accompanied by an increase in diacylglycerol acylation. Moreover, the CE increment was associated with higher activity of acyl-CoA:cholesterol acyltransferase. Interestingly, α-syn overexpression increased cholesterol lysosomal accumulation. We observed that sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 were differentially regulated by α-syn overexpression. The latter gave rise to a reduction in SREBP-1 nuclear translocation and consequently in fatty acid synthase expression, whereas it produced an increase in SREBP-2 nuclear localization. Surprisingly, and despite increased cholesterol levels, SREBP-2 downstream genes related to cholesterolgenesis were not upregulated as expected. Notably, phospholipid (PL) levels were diminished in cells overexpressing α-syn. This decrease was related to the activation of phospholipase A2 (PLA2) with a concomitant imbalance of the PL deacylation-acylation cycle. Fatty acids released from PLs by iPLA2 and cPLA2 action were esterified into TAGs, thus promoting a biological response to α-syn overexpression with uncompromised cell viability. When the described steady-state was disturbed under conditions favoring higher levels of α-syn, the response was an enhanced LD accumulation, this imbalance ultimately leading to neuronal death.

Crosstalk between sterol and neutral lipid metabolism in the alga Haematococcus pluvialis exposed to light stress.

The presence, biosynthesis and functional role of sterols in the green microalga Haematococcus pluvialis remain poorly understood. In this work we studied the effect of high-light (HL) stress on sterol synthesis in H. pluvialis UTEX 2505 cells. HL stress induced the synthesis of sterols in parallel with that of triacylglycerides (TAG), giving rise to the synthesis of cholesterol over that of phytosterols. Blockage of the carotenogenic 1-deoxy-D-xylulose 5-phosphate (MEP) pathway is shown to be involved in HL-induced sterol synthesis. In addition, high irradiance exposure induced MEP- and fatty acid (FA)-biosynthetic transcripts. The pharmacological inhibition of these pathways suggests a possible feedback regulation of sterol and FA homeostasis. Finally, both lipid classes proved crucial to the adequate photosynthetic performance of H. pluvialis grown under HL intensity stress. Our findings reveal new insights into H. pluvialis lipid metabolism that contribute to the development of value-added bioproducts from microalgae.

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease.

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.

Incidencia e importancia pronóstica del deterioro de la función renal en pacientes hospitalizados con insuficiencia cardiaca / Deterioration of kidney function as a risk factor for mortality among patients hospitalized for heart failure

Background: Acute deterioration of kidney function among patients admitted to the hospital for cardiac failure is associated with an increased mortality. Aim: To investigate the association between deterioration of kidney function and mortality among patients hospitalized for cardiac failure. Material and Methods: Patients admitted for decompensated cardiac failure to 14 Chilean hospitals between 2002 and 2009 were incorporated to the study. Clinical and laboratory features were registered. Serum creatinine values on admission and discharge were determined. Hospital and long term mortality was determined requesting death certificates to the National Identification Service at the end of follow up, lasting 635 ± 581 days. Results: One thousand sixty four patients were incorporated and 1100, aged 68 ± 13 years (45% females) had information about renal function. Seventy seven percent were hypertensive and 36% were diabetic. Mean ejection fraction was 41 ± 18% and 34% had an ejection fraction over 50%. Mean admission creatinine was 1.7 ± 1.6 mg/dl and 19% had a creatinine over 2 mg/dl. Serum creatinine increased more than 0.5 mg/dl during hospitalization in 9% of general patients and in 11% of diabetics. The increase in creatinine was associated with a higher risk of hospital mortality (odds ratio (OR) 12.9, 95% confidence intervals (CI) 6.7-27.6) and long term mortality (OR 2.1, 95% CI 1.6-3). Conclusions: The deterioration of renal function during hospitalization of patients with heart failure is a risk factor for hospital and long term mortality.

Extracellular ATP regulates FoxO family of transcription factors and cell cycle progression through PI3K/Akt in MCF-7 cells.

BACKGROUND: Forkhead Box-O (FoxO) transcription factors regulate the expression of many genes involved in suppression. Released nucleotides can regulate intracellular signaling pathways through membrane-bound purinergic receptors, to promote or prevent malignant cell transformation. We studied the role of extracellular ATP in the modulation of Forkhead Box O (FoxO) transcription factors and of cell cycle progression in MCF-7 breast cancer cells. METHODS: Western blot analysis, cell transfections with siRNA against Akt, immunocytochemistry, subcellular fractionation studies and flow cytometry analysis were performed. RESULTS: ATP induced the phosphorylation of FoxO1/3a at threonine 24/32, whereas reduced the expression of FoxO1. In addition, ATP increased the expression of the cyclins D1 and D3 and down-regulated the cell cycle inhibitory proteins p21Cip1 and p27Kip1. The use of the phosphatidylinositol 3 kinase (PI3K) inhibitor, Ly294002, and/or of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. ATP induced the translocation of FoxO3a from the nucleus to the cytoplasm. Also, ATP increased the number of cells in the S phase of cell cycle; this effect was reverted by the use of Ly294002 and the proteasome inhibitor bortezomib. CONCLUSION: Extracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression through the PI3K/Akt pathway in MCF-7 cells. GENERAL SIGNIFICANCE: These findings provide new molecular basis for further understanding the mechanisms involved in ATP signal transduction in breast cancer cells, and should be considered for the development of effective breast cancer therapeutic strategies.

[Deterioration of kidney function as a risk factor for mortality among patients hospitalized for heart failure]. / Incidencia e importancia pronóstica del deterioro de la función renal en pacientes hospitalizados con insuficiencia cardiaca.

BACKGROUND: Acute deterioration of kidney function among patients admitted to the hospital for cardiac failure is associated with an increased mortality. AIM: To investigate the association between deterioration of kidney function and mortality among patients hospitalized for cardiac failure. MATERIAL AND METHODS: Patients admitted for decompensated cardiac failure to 14 Chilean hospitals between 2002 and 2009 were incorporated to the study. Clinical and laboratory features were registered. Serum creatinine values on admission and discharge were determined. Hospital and long term mortality was determined requesting death certificates to the National Identification Service at the end of follow up, lasting 635 ± 581 days. RESULTS: One thousand sixty four patients were incorporated and 1100, aged 68 ± 13 years (45% females) had information about renal function. Seventy seven percent were hypertensive and 36% were diabetic. Mean ejection fraction was 41 ± 18% and 34% had an ejection fraction over 50%. Mean admission creatinine was 1.7 ± 1.6 mg/dl and 19% had a creatinine over 2 mg/dl. Serum creatinine increased more than 0.5 mg/dl during hospitalization in 9% of general patients and in 11% of diabetics. The increase in creatinine was associated with a higher risk of hospital mortality (odds ratio (OR) 12.9, 95% confidence intervals (CI) 6.7-27.6) and long term mortality (OR 2.1, 95% CI 1.6-3). CONCLUSIONS: The deterioration of renal function during hospitalization of patients with heart failure is a risk factor for hospital and long term mortality.

Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function.

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation.

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 µM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 µM, 2.00 ± 0.2 µM and 2.4 ± 0.4 µM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na3VO4 and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.

ATP stimulates the proliferation of MCF-7 cells through the PI3K/Akt signaling pathway.

We studied the modulation of the PI3K/Akt signaling pathway by ATP in MCF-7 cells. Western blot analysis showed that ATP stimulated the phosphorylation of Akt in a dose- and time-dependent manner. Akt phosphorylation in response to nucleotides followed the potency order ATP=UTP=ATPgammaS>>ADP=UDP>ADPbetaS=adenosine, suggesting participation of P2Y(2/4) receptors. Inhibitors of PI3K, PLC, PKC and Src or Src antisense oligonucleotides prevented ATP-induced phosphorylation of Akt. Incubation of cells with 2-APB or in a nominally Ca(2+)-free medium plus EGTA showed that Akt phosphorylation by ATP depends on intracellular calcium release but is independent of calcium influx. The PI3K inhibitor was not effective in reducing MAPKs phosphorylation by ATP. ATP and UTP stimulated MCF-7 cell proliferation, effect that was inhibited by PI3K, PLC, PKC, Src and MAPKs inhibitors. These findings suggest that ATP modulation of P2Y(2/4) receptors increases MCF-7 cell proliferation by activation of the PI3K/Akt signaling pathway through PLC/IP(3)/Ca(2+), PKC and Src.

ATP modulates transcription factors through P2Y2 and P2Y4 receptors via PKC/MAPKs and PKC/Src pathways in MCF-7 cells.

In this work, we studied the involvement of PKC and Src in the phosphorylation of ERK1/2, p38 and JNK1 MAPKs and in the modulation of ATF-1, c-Fos, c-Jun and Jun D transcription factors by ATP in MCF-7 breast cancer cells. RT-PCR studies and nucleotide sequence analysis confirmed first the expression of P2Y(2)- and P2Y(4)-receptor subtypes. The use of specific inhibitors and Src antisense oligonucleotides showed that PKC, but not Src, plays a role in the phosphorylation of MAPKs by ATP. ATP stimulated the expression of c-Fos and the phosphorylation c-Jun, Jun D and ATF-1. PKC and Src only participated in c-Fos induction and in ATF-1 phosphorylation. Pharmacological inhibition of MAPKs demonstrated that c-Fos induction and phosphorylation of c-Jun and Jun D, but not of ATF-1, depend on MAPK activation. These results suggest that stimulation of P2Y(2) and P2Y(4) receptors by ATP modulates transcription factors through PKC/MAPKs and PKC/Src pathways in MCF-7 cells.

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line.

BACKGROUND: ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell. METHODS AND RESULTS: We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 microM ATP. Moreover, ATPgammaS, UTP, and UDP but not ADP or ADPbetaS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 and P2Y6 receptor subtypes. RT-PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca2+ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR. GENERAL SIGNIFICANCE: These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.

ATP modulation of mitogen activated protein kinases and intracellular Ca2+ in breast cancer (MCF-7) cells.

In the breast tumor cell line MCF-7, extracellular nucleotides induce transient elevations in intracellular calcium concentration ([Ca(2+)](i)). In this study we show that stimulation with ATP or UTP sensitizes MCF-7 cells to mechanical stress leading to an additional transient Ca(2+) influx. ATP> or =ATPgamma-S> or =UTP>>>ADP=ADPbeta-S elevate [Ca(2+)](i), proving the presence of P2Y(2)/P2Y(4) purinergic receptor subtypes. In addition, cell stimulation with ATP, ATPgamma-S or UTP but not ADPbeta-S induced the phosphorylation of ERK1/2, p38 and JNK1/2 mitogen activated protein kinases (MAPKs). The use of Gd(3+), La(3+) or a Ca(2+)-free medium, inhibited ATP-dependent stress activated Ca(2+) (SAC) influx, but had no effect on MAPK phosphorylation. ATP-induced activation of MAPKs was diminished by two PI-PLC inhibitors and an IP(3) receptor antagonist. These results evidence an ATP-sensitive SAC influx in MCF-7 cells and indicate that phosphorylation of MAPKs by ATP is dependent on PI-PLC/IP(3)/Ca(2+)(i) release but independent of SAC influx in these cells, differently to other cell types.

[Emergency of vancomycin-resistant Enterococcus infections in a teaching hospital in Chile]. / Emergencia de infecciones por Enterococcus sp resistente a vancomicina en un hospital universitario en Chile.

An active surveillance of vancomycin-resistant enterococci (VRE) intestinal colonization in selected group of patients has been developed in Chile since year 2000. Nevertheless, no reports of clinical cases have been published. Aim. To describe main clinical and microbiological features of patients infected by VRE in a tertiary-level teaching Hospital. Patients and methods. Intestinal and clinical samples positive to VRE were provided by laboratory, and a retrospective analysis of potential risk factors, clinical features, treatment and outcomes was performed. Study encompassed years 2001 to 2006. Main results. 23 cases of infections were identified, all cases occurring during 2005 and 2006. Incidence rate was 0.07 and 0.09 cases per 1000 occupied bed-days, respectively. The mean age was 62.0 +/- 17 years. A significant proportion of patients had cancer (39.1%), recent surgical procedures (54.1%), were on dialysis (26.1%), or were using steroids (26.1%). Most patients had received 2 or more antimicrobial (87%), almost a third represented transfers from other hospitals and an additional 22% readmissions before 30 days of latest discharge. Patients were mainly hospitalized in the ICU (60.9%) but nearly 30% were associated exclusively to nephrological or onco-hematological wards. Clinical manifestations included bacteremia (30.4%), surgical site infections or abscesses (26.1%), urinary tract infections (26.1%) and others. . Three patients (13%) did not have symptoms. After identification was possible, all isolates were identified as E. faecium (82.6% of total), the rest as Enterococcus sp. Most strains showed intermediate susceptibility to vancomycin (66.7%). For 14 strains studied both with vancomycin and teicoplanin, , phenotype Van B was predominant (85.7%), followed by VanA (7.1%) and VanB/VanD type (7.1%). No molecular studies were performed. Fifteen patients (65.4%) received a surgical and/or medical treatment. A favorable response was observed in 80% of these cases. Eight patients were not treated (34.8%), in 2 cases because of a rapidly-fatal infection. The global risk-fatality ratio for VRE infections was 13% and increased to 42.9% in patients with bacteremia. Microbiological eradication was documented in 52.2%