Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

Multiple Sclerosis in the Mount Etna Region: Possible Role of Volcanogenic Trace Elements

Background

Trace elements have been hypothesised to be involved in the pathogenesis of Multiple Sclerosis and volcanic degassing is the major natural sources of trace elements. Both incidence of Multiple Sclerosis in Catania and volcanic activity of Mount Etna have been significantly increased during the last 30 years. Due to prevailing trade winds direction, volcanic gases from Etna summit craters are mostly blown towards the eastern and southern sectors of the volcano.

Objective

To evaluate the possible association between Multiple Sclerosis and exposure to volcanogenic trace elements.

Methods

We evaluated prevalence and incidence of Multiple Sclerosis in four communities (47,234 inhabitants) located in the eastern flank and in two communities (52,210 inhabitants) located in the western flank of Mount Etna, respectively the most and least exposed area to crater gas emissions.

Results

A higher prevalence was found in the population of the eastern flank compared to the population of the western one (137.6/100,000 versus 94.3/100,000; p-value 0.04). We found a borderline significantly higher incidence risk during the incidence study period (1980–2009) in the population of the eastern flank 4.6/100,000 (95% CI 3.1–5.9), compared with the western population 3.2/100,000 (95% CI 2.4–4.2) with a RR of 1.41 (95% CI 0.97–2.05; p-value 0.06). Incidence risks have increased over the time in both populations reaching a peak of 6.4/100,000 in the eastern flank and of 4.4/100.000 in the western flank during 2000–2009.

Conclusion

We found a higher prevalence and incidence of Multiple Sclerosis among populations living in the eastern flank of Mount Etna. According to our data a possible role of TE cannot be ruled out as possible co-factor in the MS pathogenesis. However larger epidemiological study are needed to confirm this hypothesis.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.     

Methyl jasmonate induced responses in four plant species and its effect on Spodoptera litura Fab. performance

Defensive proteins, such as polyphenol oxidase (PPO) and trypsin inhibitor (TI), are induced by herbivore wounding and exogenous methyl jasmonate application in various plant species. This study was conducted to measure induction of PPO and TI in radish, sweet pepper, tomato, and water spinach plants following herbivore wounding (I), methyl jasmonate application (M), and a combination of the two treatments (M + I). The effect of induced responses was also examined against third instar Spodoptera litura Fab. PPO activity was induced in radish by treatment I only; in sweet pepper, by treatments I and M; in tomato, by treatments I, M, and M + I; and in water spinach, by treatments M and M + I. The activity of TI was enhanced 1.2–1.4-fold in radish, sweet pepper, and tomato by M and M + I treatments, whereas in water spinach, it was enhanced 1.2-fold by all 3 treatments. The relative growth rate (RGR) of S. litura was reduced by 53% on radish plants following M treatment only. It was reduced by 37% and 42% on sweet paper plants following M and M + I treatment, respectively. RGR was significantly reduced on test tomato plants following I, M, and M + I treatments. The RGR of S. litura was unaffected on water spinach plants following any treatment. Collectively, the results of this study indicated that induction of plant defensive proteins in response to S. litura feeding or exogenous methyl jasmonate application varied among plant species, which further affected the induced plant resistance to the caterpillars.     

Analysis of short stature homeobox-containing gene (<Emphasis Type="Italic">SHOX</Emphasis>) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.     

Can the introduction of Xenopus laevis affect native amphibian populations? Reduction of reproductive occurrence in presence of the invasive species

Biological invasions are regarded as a form of global change and potential cause of biodiversity loss. Xenopus laevis is an anuran amphibian native to sub-Saharan Africa with strong invasive capacity, especially in geographic regions with a Mediterranean climate. In spite of the worldwide diffusion of X. laevis, the effective impact on local ecosystems and native amphibian populations is poorly quantified. A large population of X. laevis occurs in Sicily and our main aim of this work was to assess the consequences of introduction of this alien species on local amphibian populations. In this study we compare the occurrence of reproduction of native amphibians in ponds with and without X. laevis, and before and after the alien colonization. The results of our study shows that, when X. laevis establishes a conspicuous population in a pond system, the populations of Discoglossus pictus, Hyla intermedia and Pelophylax synklepton esculentus show clear signs of distress and the occurrence of reproduction of these native amphibians collapses. In contrast, the populations of Bufo bufo do not appear to be affected by the alien species. Since the Sicilian population of X. laevis shows a strong dispersal capacity, proportionate and quick interventions become necessary to bound the detriment to the Sicilian amphibians populations.     

Targeting intracellular calcium cycling in catecholaminergic polymorphic ventricular tachycardia: a theoretical investigation

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant arrhythmogenic disorder linked to mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin, predisposing the young to syncope and cardiac arrest. To define the role of β-adrenergic stimulation (BAS) and to identify potential therapeutic targeted sites relating to intracellular calcium cycling, we used a Luo-Rudy dynamic ventricular myocyte model incorporated with interacting Markov models of the L-type Ca(2+) channel (I(Ca,L)) and RyR2 to simulate the heterozygous state of mouse RyR2 R4496C mutation (RyR2(R4496C+/-)) comparable with CPVT patients with RyR2 R4497C mutation. Characteristically, in simulated cells, pacing at 4 Hz or faster or pacing at 2 Hz under BAS with effects equivalent to those of isoproterenol at ≥ 0.1 μM could readily induce delayed afterdepolarizations (DADs) and DAD-mediated triggered activity (TA) in RyR2(R4496C+/-) but not in the wild-type via enhancing both I(Ca,L) and sarcoplasmic reticulum (SR) Ca(2+) ATPase (I(UP)). Moreover, with the use of steady state values of isolated endocardial (Endo), mid-myocardial (M), and epicardial (Epi) cells as initial data for conducting single cell and one-dimensional strand studies, the M cell was more vulnerable for developing DADs and DAD-mediated TA than Endo and Epi cells, and the gap junction coupling represented by diffusion coefficient (D) of ≤ 0.000766*98 cm(2)/ms was required for generating DAD-mediated TA in RyR2(R4496C+/-). Whereas individual reduction of Ca(2+) release channel of SR and Na-Ca exchanger up to 50% was ineffective, 30% or more reduction of either I(Ca,L) or I(UP) could totally suppress the inducibility of arrhythmia under BAS. Of note, 15% reduction of both I(Ca,L) and I(UP) exerted a synergistic antiarrhythmic efficacy. Findings of this model study confirm that BAS facilitates induction of ventricular tachyarrhythmias via its action on intracellular Ca(2+) cycling and a pharmacological regimen capable of reducing I(Ca,L) could be an effective adjunctive to β-adrenergic blockers for suppressing ventricular tachyarrhythmias during CPVT.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

康川间白蜡种虫运输测验

白蜡虫是我国的特产,古代农民远在元朝已有饲养。在过去全国的白蜡年产量约在五万至十万担间,据1918年重庆海关报告有一万一千余担,值银七十七万余海 关两。解放前一、二十年来,由于国民党反动派的不重视和摧残,已逐渐衰落。白蜡是化学工业上的重要原料,用途很广,饲养蜡虫又是西南各省农民的主要副业,而且农民群众已积累了非常丰富的传统经验。因此,为了总结群众经验,发扬科学遗产,改进饲养技术,振兴蜡业,我们希望能引起有关方面的注意和进一步研究,除研究改进白蜡虫的经管技术外,又须注意白蜡在工业上的用途。 白蜡虫的种蜡异地繁荣,在目前已成为自然的形势,而蜡区种虫不能自给,是白蜡业发展上的基本障碍。我们通过本次测验之后,认为我国适于蜡虫(雄虫)繁育的地方很多,如四川、湖南、浙江、安徽、福建、江西、江苏等丘陵地带都可扑虫产蜡,关键问题在于改善运种方法。在目前我国交通运输业已有普遍发展的基础上,只要用航空运种即可在短时间内将种虫运送至全国蜡区。扩大蜡区,增加农民副业收入,发扬我国科学遗产,为国家创造财富,利用益虫为生产服务,在目前已完全有实现的可能。     

Hypothesis: hyperstructures regulate initiation in Escherichia coli and other bacteria

Hyperstructures or modules have been proposed to constitute a level of organisation intermediate between macromolecules and whole cells. In this model of intracellular organisation, hyperstructures compete and collaborate for existence within the membrane and cytoplasm. Those directly involved in the cell cycle include initiation, replication and division hyperstructures based on DnaA, SeqA and the 2-minute cluster, respectively. During the run-up to initiation, the mass to DNA ratio increases and, we contend, differential gene expression leads to some hyperstructures becoming more active and stable than others. This results in a drop in the diversity of hyperstructures, some of which release DnaA as they dissociate, and a DnaA-initiation hyperstructure forms. Subsequent DNA replication and cell division generate different daughter cells containing different hyperstructures. This has the advantage of increasing the phenotypic diversity of the population. In developing this model, we also invoke hyperstructures in the partitioning of origins of replication.