A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 μM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 μM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 μM. Lactate and ethanol were also accumulated at 760 μM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 μM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 μM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 μM h(-1) and K(s)=5.8 and 13.1 μM for control and bioaugmented sludges, respectively).     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.     

Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP

Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.     

SKP2 oncogene is a direct MYC target gene and MYC down-regulates p27(KIP1) through SKP2 in human leukemia cells

SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.     

Minimising mortality in endangered raptors due to power lines: the importance of spatial aggregation to optimize the application of mitigation measures

Electrocution by power lines is one of the main causes of non-natural mortality in birds of prey. In an area in central Spain, we surveyed 6304 pylons from 333 power lines to determine electrocution rates, environmental and design factors that may influence electrocution and the efficacy of mitigation measures used to minimise electrocution cases. A total of 952 electrocuted raptors, representing 14 different species, were observed. Electrocuted raptors were concentrated in certain areas and the environmental factors associated with increased electrocution events were: greater numbers of prey animals; greater vegetation cover; and shorter distance to roads. The structural elements associated with electrocutions were shorter strings of insulators, one or more phases over the crossarm, cross-shaped design and pylon function. Of the 952 carcasses found, 148 were eagles, including golden eagle (Aquila chrysaetos), Spanish imperial eagle (Aquila adalberti) and Bonelli's eagle (Aquila fasciata). Electrocuted eagles were clustered in smaller areas than other electrocuted raptors. The factors associated with increased eagle electrocution events were: pylons function, shorter strings of insulators, higher slopes surrounding the pylon, and more numerous potential prey animals. Pylons with increased string of insulators had lower raptor electrocution rates than unimproved pylons, although this technique was unsuccessful for eagles. Pylons with cable insulation showed higher electrocution rates than unimproved pylons, both for raptors and eagles, despite this is the most widely used and recommended mitigation measure in several countries. To optimize the application of mitigation measures, our results recommend the substitution of pin-type insulators to suspended ones and elongating the strings of insulators.     

Elevated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells

Background

The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

Methodology/Principal Findings

Here, we show that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefitinib efflux from cells. In addition, BCRP/ABCG2 expression correlates with poor response to gefitinib in both cancer cell lines and lung cancer patients with wtEGFR. Co-treatment with BCRP/ABCG2 inhibitors enhanced the anti-tumor activity of gefitinib.

Conclusions/Significance

Thus, BCRP/ABCG2 expression may be a predictor for poor efficacy of gefitinib treatment, and targeting BCRP/ABCG2 may broaden the use of gefitinib in patients with wtEGFR.     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Crown responses to neighbor density and species identity in a young mixed deciduous stand

Key message

Density was more important in shaping crown structure than neighbor species identity. Both species showed high crown plasticity at alternative levels, which may explain species coexistence in mixed broadleaved forests with functionally similar species.

Abstract

Understanding crown response to local competition is essential to predicting stand development in mixed stands. We analyzed data from an 8-year-old field experimental plantation mixing two species according to a crossed gradient of density and species proportion to quantify the effect of a broad range of local neighborhood conditions on the development of young trees at multiple crown levels. We used Fagus sylvatica and Acer pseudoplatanus, as two model deciduous species. They are considered functionally equivalent at the young stages, but with contrasting architectural patterns. For both species: (1) changes in density explained more of the variation on crown development than species proportion (2) much of the effect of competition was accounted for by variables at the stem level, while branch and leaf development within crowns were not directly altered by competition. Both species were able to modify their crowns at the stem level to compete with intra- and inter-specific neighbors: Acer and Fagus were taller with a highest proportion of Fagus as neighbors; Fagus displayed a lower crown base when the proportion of Fagus decreased, while Acer had a lower crown base when the proportion of Fagus around it increased. Both species showed common shapes in allometric relationships but contrasting responses at alternative crown levels. Acer exhibited broader intra-specific variation in its height–diameter relationship and in its crown length, while Fagus displayed higher individual variation of branch development and leaf area than Acer. This study demonstrates that differences in crown development strategy of each species in response to changes in local neighborhood conditions are an important factor in maintaining species coexistence in broadleaved forests and designing mixtures that persist over time.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.