Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia

Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production.     

The primary structure of iron-superoxide dismutase from Photobacterium leiognathi

The complete amino acid sequence of iron-superoxide dismutase from Photobacterium leiognathi was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus V-8 protease digests of the apoprotein. The amino acid sequence listed below is made up of 193 residues. It is the first complete sequence to be determined for an iron-superoxide dismutase. The iron-superoxide dismutase shows the same order of homology with the manganese-superoxide dismutases as these enzymes show among themselves. No homology was observed with the copper/zinc-containing class of superoxide dismutases. Ala-Phe-Glu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-Met-Asn-Ala-Leu-Glu-Pro-His-Ile- Ser-Gln-Glu-Thr-Leu-Glu-Tyr-His-Tyr-Gly-Lys-His-His-Asn-Thr-Tyr-Val-Val- Lys-Leu-Asn-Gly-Leu-Val-Glu-Gly-Thr-Glu-Leu-Ala-Glu-Lys-Ser-Leu-Glu-Glu- Ile-Ile-Lys-Thr-Ser-Thr-Gly-Gly-Val-Phe-Asn-Asn-Ala-Ala-Gln-Val-Trp-Asn- His-Thr-Phe-Tyr-Trp-Asn-Cys-Leu-Ala-Pro-Asn-Ala-Gly-Gly-Glu-Pro-Thr-Gly- Glu-Val-Ala-Ala-Ala-Ile-Glu-Lys-Ala-Phe-Gly-Ser-Phe-Ala-Glu-Phe-Lys-Ala- Lys-Phe-Thr-Asp-Ser-Ala-Ile-Asn-Asn-Phe-Gly-Ser-Ser-Trp-Thr-Trp-Leu-Val- Lys-Asn-Ala-Asn-Gly-Ser-Leu-Ala-Ile-Val-Asn-Thr-Ser-Asn-Ala-Gly-Cys-Pro- Ile-Thr-Glu-Glu-Gly-Val-Thr-Pro-Leu-Leu-Thr-Val-Asp-Leu-Trp-Glu-His-Ala- Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Leu-Arg-Pro-Ser-Tyr-Met-Asp-Gly-Phe-Trp-Ala- Leu-Val-Asn-Trp-Asp-Phe-Val-Ser-Lys-Asn-Leu-Ala-Ala.     

Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein–protein interactions in relation to subcellular localization

The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein–protein interactions and post-translational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein–protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al. in J Proteome Res 11:3996–4012, 2012). We first analyzed the colocalization of PKCζ and Bcl10 proteins through classical immunofluorescent colocalization analysis. On the basis of these results, we developed a novel imaging approach combining immunofluorescence (IF) techniques with in situ PLA to identify the PKCζ·Bcl10 complexes at the level of a specific subcellular compartment, i.e., the nuclear envelope (NE). By this means, we could show that the amount of PKCζ·Bcl10 complexes localized at the NE of C4-I cells during proliferation or after treatment with VP-16 closely corresponded to our previous purely biochemical results. Hence, the present findings demonstrate that the combination of in situ PLA with classical IF detection is a novel powerful analytical tool allowing to morphologically demonstrate new specific protein–protein interactions at level of subcellular organelles, the complexes functions of which can next be clarified through proteomic/biochemical approaches.     

Polyelectrolytic aspects of the titration curve. pH-induced conformational transition of poly(L-glutamic acid): the semi-flexible model

The cooperative conformational transition of poly(L-glutamic acid) induced by pH is monitored by the titration curves from literature. The polyelectrolytic approach described in the preceding article (A. Cesàro, S. Paoletti and J.C. Benegas, Biophys. Chem. 39 (1991) 1) is used to fit the experimental curves under various conditions of ionic strength and temperature, with the sole assumption that each polymeric state is characterized by a proper conformational flexibility. The helix-coil transition of the system becomes molecularly defined by the balance between the non-ionic conformational energy and the repulsive electrostatic energy of the two forms. Implications of the results of the theoretical model on the energetics of the cooperative order-disorder transition are discussed.     

Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.     

Associations between Genetic Polymorphisms in IL-33, IL1R1 and Risk for Inflammatory Bowel Disease

Background

Recent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients.

Methods

We evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn’s disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed.

Results

Significant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, P<0.015). By combining homo- and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. Upon genotype-phenotype evaluation, an increased frequency of extensive colitis in adult UC (P = 0.019) and in steroid-responsive pediatric patients (P = 0.024) carrying the IL-33 rs3939286 risk genotype, was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased.

Conclusions

Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.     

Competition Experiments on Alien Weeds with Crops: Lessons for Measuring Plant Invasion Impact?

Can we quantify the impact of invasive species? Here we use the per-plant competitiveness of alien weeds on crops as a model of invasive species impact in general. We reviewed 97 weed–crop competition experiments in 32 papers that included 30 alien weed and 14 crop species. The majority (68.92%) were randomised block designs where the alien weed had been either added (additive experiments) or removed (removal experiments). We propose using the relative competition index to estimate the effect of alien species in all systems, specifying in each case the density and proportion of alien and native plants essayed. We found that the impact of the weed cannot be considered independently of the crop and, thus, we should be cautious in ranking weed species according to their competition effect. A similar situation can be postulated for alien plants interfering with native species. Invaded communities are not random assemblages, and researchers tend to study the most competitive alien plants. We also found that the effect of the weed on crop yield depends on the duration of the interference and the life-history stage of the weed–crop system at which the interaction takes place. We were not able to conduct a more rigorous comparative analysis of the impacts, such as a meta-analysis. To do this would require some measure of the variation of the competition effect such as standard deviation or standard error, which we found are almost never reported.