Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial

Objective To assess the incidence, cofactors, and excess risk of development of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis, attributable to tamoxifen in women.Design Prospective, randomised, double blind, placebo controlled trial.Setting and participants 5408 healthy women who had had hysterectomies, recruited into the Italian tamoxifen chemoprevention trial from 58 centres in Italy.Intervention Women were randomly assigned to receive tamoxifen (20 mg daily) or placebo for five years.Main outcome measure Development of non-alcoholic fatty liver disease in all women with normal baseline liver function who showed at least two elevations of alanine aminotransferase (≥ 1.5 times upper limit of normal) over a six month period.Results During follow up, 64 women met the predefined criteria: 12 tested positive for hepatitis C virus, and the remaining 52 were suspected of having developed non-alcoholic fatty liver disease (34 tamoxifen, 18 placebo)—hazard ratio = 2.0 (95% confidence interval 1.1 to 3.5; P = 0.04). In all 52 women ultrasonography confirmed the presence of fatty liver. Other factors associated with the development of non-alcoholic fatty liver disease included overweight (2.4, 1.2 to 4.8), obesity (3.6, 1.7 to 7.6), hypercholesterolaemia (3.4, 1.4 to 7.8), and arterial hypertension (2.0, 1.0 to 3.8). Twenty women had liver biopsies: 15 were diagnosed as having mild to moderate steatohepatitis (12 tamoxifen, 3 placebo), and five had fatty liver alone (1 tamoxifen, 4 placebo). No clinical, biochemical, ultrasonic, or histological signs suggestive of progression to cirrhosis were observed after a median follow up of 8.7 years.Conclusions Tamoxifen was associated with higher risk of development of non-alcoholic steatohepatitis only in overweight and obese women with features of metabolic syndrome, but the disease, in both the tamoxifen and the placebo group, after 10 years of follow up seems to be indolent.     

The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae

The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation. We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast. Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate. Under high osmolarity conditions, when the Fps1p channel is closed, wild-type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Delta mutant. Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts. Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite. We also show by direct transport assays that arsenite uptake is mediated by Fps1p. Yeast cells appear to control the Fps1p-mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition. To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.     

Early changes in adenosine A1 receptors in cerebral cortex slices submitted to in vitro ischemia

The effects of brain ischemia on the maximum binding capacity (B_max) and affinity (K_d) of A₁ receptors were studied in the rat cerebral cortex, with an in vitro approach. The results were correlated with changes in ³H-adenosine release, studied under identical experimental conditions. Fifteen minutes of in vitro ‘ischemia’ (hypoxic, glucose-free medium) induced a significant increase in both B_max (2398±132 fmol/mg protein, 151% of the control, P<0.05) and in K_d (2.43±0.12 nM, 161% of the control, P<0.01). At the same time, an increase in tritium efflux from [³H]-adenosine labeled cerebral cortex slices to 324% of the control was observed. A trend toward normalization was evident 5–15 min after ‘reoxygenation’ (restoring normal medium), but the binding parameters were still altered after 60 min (B_max 2110±82 fmol/mg protein, K_d 2.26±0.14 nM, P<0.01 vs the corresponding control) as was adenosine release (196% of the control). These findings suggest that the increased availability of adenosine and its receptors may be a defense mechanism against ischemic injury, while the reduced affinity of A₁ receptors, possibly due to desensitization, may be a sign of ischemia-induced cellular damage.     

Coupling of RNA polymerase III assembly to cell cycle progression in Saccharomyces cerevisiae

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.     

The LA Loop as an Important Regulatory Element of the HtrA (DegP) Protease from Escherichia coli: STRUCTURAL AND FUNCTIONAL STUDIES*

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA′ (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.     

Mitogenomes of Polar Bodies and Corresponding Oocytes

The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of ‘normality’.     

Phosphorylation Modulates Clearance of Alpha-Synuclein Inclusions in a Yeast Model of Parkinson's Disease

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson''s disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.