Analysis of hepatitis C virus RNA dimerization and core-RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.     

Loss of the ecdysteroid-inducible E75A orphan nuclear receptor uncouples molting from metamorphosis in Drosophila

Isoform-specific null mutations were used to define the functions of three orphan members of the nuclear receptor superfamily, E75A, E75B, and E75C, encoded by the E75 early ecdysteroid-inducible gene. E75B mutants are viable and fertile, while E75C mutants die as adults. In contrast, E75A mutants have a reduced ecdysteroid titer during larval development, resulting in developmental delays, developmental arrests, and molting defects. Remarkably, some E75A mutant second instar larvae display a heterochronic phenotype in which they induce genes specific to the third instar and pupariate without undergoing a molt. We propose that ecdysteroid-induced E75A expression defines a feed-forward pathway that amplifies or maintains the ecdysteroid titer during larval development, ensuring proper temporal progression through the life cycle.     

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.     

Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects.

The exhaled breath condensate (EBC) method represents a new, noninvasive way to detect inflammatory and metabolic markers in the fluid that covers the airways [epithelial lining fluid (ELF)]. However, respiratory droplets represent only a very small and variable fraction of the EBC, most (approximately 99.99%) of which is water vapor. Our objective was to show that ELF concentrations could be calculated from EBC values by using any of three dilutional indicators (urea, total cations, and conductivity) in nine normal and nine chronic obstructive lung disease (COPD) subjects. EBC concentrations of Na(+), K(+), Ca(2+), Mg(2+), total cations, urea, and conductivity varied over a 10-fold range among individuals, but concentrations of these constituents (except Ca(2+)) remained well correlated (r(2) = 0.44-0.83, P < 0.001). Dilution (D) of respiratory droplets in water vapor was calculated by dividing plasma concentrations of the dilutional indicators by EBC concentrations. Estimates of D were not significantly different among these indicators, and urea D averaged 10,800 +/- 2,100 (SE) in normal and 12,600 +/- 3,300 in COPD subjects. Although calculated Na(+) concentrations in the ELF were less than one-half those in plasma, and concentrations of K(+), Ca(2+), and Mg(2+) exceeded those in plasma, total cation concentrations in ELF were not significantly different from those in plasma, indicating that ELF is isotonic in both normal and COPD subjects. EBC amylase concentrations (measured with an ultrasensitive procedure) indicated that saliva represented <10% of the respiratory (ELF) droplets in all but three samples. Dilutional and salivary markers are essential for interpretation of EBC studies.     

Association of selected SNP with carcass and taste panel assessed meat quality traits in a commercial population of Aberdeen Angus-sired beef cattle

Background

The purpose of this study was to evaluate the effects of eight single nucleotide polymorphisms (SNP), previously associated with meat and milk quality traits in cattle, in a population of 443 commercial Aberdeen Angus-cross beef cattle. The eight SNP, which were located within five genes: μ-calpain (CAPN1), calpastatin (CAST), leptin (LEP), growth hormone receptor (GHR) and acylCoA:diacylglycerol acyltransferase 1 (DGAT1), are included in various commercial tests for tenderness, fatness, carcass composition and milk yield/quality.

Methods

A total of 27 traits were examined, 19 relating to carcass quality, such as carcass weight and fatness, one mechanical measure of tenderness, and the remaining seven were sensory traits, such as flavour and tenderness, assessed by a taste panel.

Results

An SNP in the CAPN1 gene, CAPN316, was significantly associated with tenderness measured by both the tenderometer and the taste panel as well as the weight of the hindquarter, where animals inheriting the CC genotype had more tender meat and heavier hindquarters. An SNP in the leptin gene, UASMS2, significantly affected overall liking, where animals with the TT genotype were assigned higher scores by the panellists. The SNP in the GHR gene was significantly associated with odour, where animals inheriting the AA genotype produced steaks with an intense odour when compared with the other genotypes. Finally, the SNP in the DGAT1 gene was associated with sirloin weight after maturation and fat depth surrounding the sirloin, with animals inheriting the AA genotype having heavier sirloins and more fat.

Conclusion

The results of this study confirm some previously documented associations. Furthermore, novel associations have been identified which, following validation in other populations, could be incorporated into breeding programmes to improve meat quality.     

Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.     

Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaH(o), DeltaS(o), and melting temperature (T(M)) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaH(o), DeltaDeltaS(o), DeltaDelta, and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaH(o) and DeltaDeltaS(o) is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaH(o), DeltaDeltaS(o), and DeltaDelta parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MX(L) and 5' X(L)N, where M, N, and X = A, C, G, or T and X(L) represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T(M) estimates accurate to within 2 degrees C for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.