Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value < 1 × 10⁻⁵) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p < 1 × 10⁻⁵). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10⁻¹⁰, odds ratio 1.15 [1.10–1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04–1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression.     

Over-expression of the ��-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content

Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS-mediated TNF-α and IL-6 production through inhibiting NF-κB activation and MAPK pathway

Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.     

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.     

Reduced levels of cytochrome bf complex in transgenic tobacco leads to marked photochemical reduction of the plastoquinone pool,without significant change in acclimation to irradiance

Tobacco transgenics with decreased amounts of the FeS apoprotein were generated using an antisense RNA construct targeted against the nuclear-encoded Rieske FeS protein of cytochrome bf complex [Price et al. (1995) Aust J Plant Physiol 22: 285–297]. FeS phenotypes ranging from intermediate to low were obtained which had 69% and 26% of the Rieske FeS protein of wild type. Similar reductions in the other subunits of cytochrome bf complex, cytochrome f, cytochrome b₅₆₃and the 17 kDa subunit, were demonstrated in the thylakoids of intermediate and low FeS phenotypes. Confirmation that the levels of assembled cytochrome bf in leaves matched the levels of the FeS protein was demonstrated by laser flash-induced redox absorbance changes in leaves, with the extents of cytochrome f oxidation and cytochrome b₅₆₃reduction being equivalent to the decreased amounts of the subunits in isolated thylakoids of the antisense plants. Despite greatly enhanced photochemical reduction of Q_Aand the plastoquinone pool in the antisense plants, light acclimation of the FeS phenotypes to irradiance did not occur. Furthermore, the state 1–state 2 transitions were identical in wild type and antisense plants. Our results suggest that neither Q_Anor the plastoquinone pool acts alone in either the redox control of gene expression or the regulation of light energy distribution between the photosystems. We suggest rather that reduced plastoquinone acting at the inner Q_psite of cytochrome bf complex is involved in molecular redox signalling.     

Oxygen exchange in leaves in the light

Photosynthetic O₂ production and photorespiratory O₂ uptake were measured using isotopic techniques, in the C₃ species Hirschfeldia incana Lowe., Helianthus annuus L., and Phaseolus vulgaris L. At high CO₂ and normal O₂, O₂ production increased linearly with light intensity. At low O₂ or low CO₂, O₂ production was suppressed, indicating that increased concentrations of both O₂ and CO₂ can stimulate O₂ production. At the CO₂ compensation point, O₂ uptake equaled O₂ production over a wide range of O₂ concentrations. O₂ uptake increased with light intensity and O₂ concentration. At low light intensities, O₂ uptake was suppressed by increased CO₂ concentrations so that O₂ uptake at 1,000 microliters per liter CO₂ was 28 to 35% of the uptake at the CO₂ compensation point. At high light intensities, O₂ uptake was stimulated by low concentrations of CO₂ and suppressed by higher concentrations of CO₂. O₂ uptake at high light intensity and 1000 microliters per liter CO₂ was 75% or more of the rate of O₂ uptake at the compensation point. The response of O₂ uptake to light intensity extrapolated to zero in darkness, suggesting that O₂ uptake via dark respiration may be suppressed in the light. The response of O₂ uptake to O₂ concentration saturated at about 30% O₂ in high light and at a lower O₂ concentration in low light. O₂ uptake was also observed with the C₄ plant Amaranthus edulis; the rate of uptake at the CO₂ compensation point was 20% of that observed at the same light intensity with the C₃ species, and this rate was not influenced by the CO₂ concentration. The results are discussed and interpreted in terms of the ribulose-1,5-bisphosphate oxygenase reaction, the associated metabolism of the photorespiratory pathway, and direct photosynthetic reduction of O₂.     

An immunostimulatory substance in the ascites fluid of patients with cancer metastatic to peritoneum.

The ascitic fluids from patients with cancer metastatic to the peritoneum contain a factor(s) which stimulates the primary antibody response to sheep red blood cells (SRBC) in vitro. This enhancement is manifested by an increase in the number of plaque-forming cells per culture and a slight increase in plaque size. This factor has a molecular weight in the range 30,000–100,000 as determined by Sephadex gel filtration. The factor, which we have called “stimulatory factor” (SF), will completely replace the requirement for fetal calf serum in the Mishell-Dutton type of assay. Enhancement of the antibody response is most apparent at suboptimal culture conditions. SF does not increase the number of plaque-forming cells to the T-independent antigen Escherichia coli but there is a marked increase in the size of the plaques produced to the lipopolysaccharide using coated SRBC as targets. The stimulation induced by this factor is not due to endotoxin contamination since endotoxin is heat stable and the SF is heat inactivated at 80 °C for 10 min. In addition endotoxin does not act in a manner similar to SF. Thus, the SF appears to influence both T and B cells. With thymus-dependent antigen the factor results in increased numbers of antibody cells being generated; with thymus-independent antigen the factor results in increased quantity of antibody being produced.     

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.