Association of polymorphic markers I/D of gene ACE and A1166C of gene AT2R1 with ischemic chronic heart failure in the Russian and Tatar populations of Bashkortostan Republic

Polymerase chain reaction was used to study the association of polymorphic markers I/D of the angiotensin-converting enzyme gene (ACE) and A1166C of the angiotensin II type 1 receptor gene (AT2R1) with chronic heart failure (CHF) in Russian and Tatar patients that had had myocardial infarction. In Russian patients aged 50 years or younger that had had macrofocal myocardial infarction, the CC genotype of the A1166C polymorphic marker of gene AT2R1 was associated with an increased risk of CHF (OR = 11.36). Genotype DD and allele D of the I/D polymorphic marker of gene ACE were associated with a more severe CHF (functional class III–IV) in Russian patients (OR = 3.50 and 2.06). In Tatar patients, polymorphic markers I/D of gene ACE and A1166C of gene AT2R1 were not associated with CHF.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Association of polymorphic markers I/D of gene ACE and A1166C of gene AT2R1 with ischemic chronic heart failure in the Russian and Tatar populations of Bashkortostan Republic

Polymerase chain reaction was used to study the association of polymorphic markers I/D of the angiotensin-converting enzyme gene (ACE) and A1166C of the angiotensin II type 1 receptor gene (AT2R1) with chronic heart failure (CHF) in Russian and Tatar patients that had had myocardial infarction. In Russian patients aged 50 years or younger that had had macrofocal myocardial infarction, the CC genotype of the A1166C polymorphic marker of gene AT2R1 was associated with an increased risk of CHF (OR = 11.36). Genotype DD and allele D of the I/D polymorphic marker of gene ACE were associated with a more severe CHF (functional class III-IV) in Russian patients (OR = 3.50 and 2.06). In Tatar patients, polymorphic markers I/D of gene ACE and A1166C of gene AT2R1 were not associated with CHF.     

Potassium ions are required for nucleotide-induced closure of gyrase N-gate

DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA by a strand passage mechanism that requires coordinated opening and closing of three protein interfaces, the N-, DNA-, and C-gates. ATP binding to the GyrB subunits of gyrase causes dimerization and N-gate closure. The closure of the N-gate is a key step in the gyrase catalytic cycle, as it captures the DNA segment to be transported and poises gyrase toward strand passage. We show here that K(+) ions are required for DNA supercoiling but are dispensable for ATP-independent DNA relaxation. Although DNA binding, distortion, wrapping, and DNA-induced narrowing of the N-gate occur in the absence of K(+), nucleotide-induced N-gate closure depends on their presence. Our results provide evidence that K(+) ions relay small conformational changes in the nucleotide-binding pocket to the formation of a tight dimer interface at the N-gate by connecting regions from both GyrB monomers and suggest an important role for K(+) in synchronization of N-gate closure and DNA-gate opening.     

The cost-effectiveness of reclassification sampling for prevalence estimation

Background

Typically, a two-phase (double) sampling strategy is employed when classifications are subject to error and there is a gold standard (perfect) classifier available. Two-phase sampling involves classifying the entire sample with an imperfect classifier, and a subset of the sample with the gold-standard.

Methodology/Principal Findings

In this paper we consider an alternative strategy termed reclassification sampling, which involves classifying individuals using the imperfect classifier more than one time. Estimates of sensitivity, specificity and prevalence are provided for reclassification sampling, when either one or two binary classifications of each individual using the imperfect classifier are available. Robustness of estimates and design decisions to model assumptions are considered. Software is provided to compute estimates and provide advice on the optimal sampling strategy.

Conclusions/Significance

Reclassification sampling is shown to be cost-effective (lower standard error of estimates for the same cost) for estimating prevalence as compared to two-phase sampling in many practical situations.     

Universal Internucleotide Statistics in Full Genomes: A Footprint of the DNA Structure and Packaging?

Uncovering the fundamental laws that govern the complex DNA structural organization remains challenging and is largely based upon reconstructions from the primary nucleotide sequences. Here we investigate the distributions of the internucleotide intervals and their persistence properties in complete genomes of various organisms from Archaea and Bacteria to H. Sapiens aiming to reveal the manifestation of the universal DNA architecture. We find that in all considered organisms the internucleotide interval distributions exhibit the same -exponential form. While in prokaryotes a single -exponential function makes the best fit, in eukaryotes the PDF contains additionally a second -exponential, which in the human genome makes a perfect approximation over nearly 10 decades. We suggest that this functional form is a footprint of the heterogeneous DNA structure, where the first -exponential reflects the universal helical pitch that appears both in pro- and eukaryotic DNA, while the second -exponential is a specific marker of the large-scale eukaryotic DNA organization.     

eIF4B,eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle

Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5''-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.     

Molecular architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase

The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.