A Comparison of Protein and mRNA Expression during Development of the Soil Dwelling Prokaryote (S. coelicolor)

In this paper, correlation analysis of protein and mRNA levels in the soil dwelling bacteria Streptomyces coelicolor (S. coelicolor M145) is presented during development of the population as it grew in liquid medium using three biological and two technical replicates, measured during exponential growth, and its entry into the stationary phase. The proteome synthesis time series are compared with the gene expression time series measured previously under identical experimental conditions. Results reveal that about one third of protein/mRNA synthesis profiles are well correlated while another third are correlated negatively. Functional analysis of the highly correlated groups is presented. Based on numerical simulation, the negative correlation between protein and mRNA is shown to be caused by the difference between the rate of translation and protein degradation.     

DPT tautomerization of the long A∙A* Watson-Crick base pair formed by the amino and imino tautomers of adenine: combined QM and QTAIM investigation

Combining quantum-mechanical (QM) calculations with quantum theory of atoms in molecules (QTAIM) and using the methodology of sweeps of the energetic, electron-topological, geometric and polar parameters, which describe the course of the tautomerization along the intrinsic reaction coordinate (IRC), we showed for the first time that the biologically important A?A* base pair (C_s symmetry) formed by the amino and imino tautomers of adenine (A) tautomerizes via asynchronous concerted double proton transfer (DPT) through a transition state (TS), which is the A⁺?A^? zwitterion with the separated charge, with C_s symmetry. The nine key points, which can be considered as electron-topological “fingerprints” of the asynchronous concerted A?A*?A*?A tautomerization process via the DPT, were detected and completely investigated along the IRC of the A?A*?A*?A tautomerization. Based on the sweeps of the H-bond energies, it was found that intermolecular antiparallel N6Н?N6 (7.01 kcal mol^?1) and N1H?N1 (6.88 kcal mol^?1) H-bonds are significantly cooperative and mutually reinforce each other. It was shown for the first time that the A?A*?A*?A tautomerization is assisted by the third C2H?HC2 dihydrogen bond (DHB), which, in contrast to the two others N6H?N6 and N1H?N1 H-bonds, exists within the IRC range from ?2.92 to 2.92 Å. The DHB cooperatively strengthens, reaching its maximum energy 0.42 kcal mol^?1 at IRC?=??0.52 Å and minimum energy 0.25 kcal mol^?1 at IRC?=??2.92 Å, and is accompanied by strengthening of the two other aforementioned classical H-bonds. We established that the C2H?HC2 DHB completely satisfies the electron-topological criteria for H-bonding, in particular Bader’s and all eight “two-molecule” Koch and Popelier’s criteria. The positive value of the Grunenberg’s compliance constant (5.203 Å/mdyn) at the TS_A?A*?A*?A proves that the C2H?HC2 DHB is a stabilizing interaction. NBO analysis predicts transfer of charge from σ(C2–H) bonding orbital to σ*(H–C2) anti-bonding orbital; at this point, the stabilization energy E⁽²⁾ is equal to 0.19 kcal mol^?1 at the TS_A?A*?A*?A.     

OXIDIZED LIPIDS DID NOT REDUCE LIFESPAN IN THE FRUIT FLY,Drosophila melanogaster

Aging is often associated with accumulation of oxidative damage in proteins and lipids. However, some studies do not support this view, raising the question of whether high levels of oxidative damage are associated with lifespan. In the current investigation, Drosophila melanogaster flies were kept on diets with 2 or 10% of either glucose or fructose. The lifespan, fecundity, and feeding as well as amounts of protein carbonyls (PC) and lipid peroxides (LOOH), activities of superoxide dismutase (SOD), catalase, glutathione‐S‐transferase (GST), and glutathione reductase activity of thioredoxin reductase (TrxR) were measured in “young” (10‐day old) and “aged” (50‐day old) flies. Flies maintained on diets with 10% carbohydrate lived longer than those on the 2% diets. However, neither lifespan nor fecundity was affected by the type of carbohydrate. The amount of PC was unaffected by diet and age, whereas flies fed on diets with 10% carbohydrate had about fivefold higher amounts of LOOH compared to flies maintained on the 2% carbohydrate diets. Catalase activity was significantly lower in flies fed on diets with 10% carbohydrates compared to flies on 2% carbohydrate diets. The activities of SOD, GST, and TrxR were not affected by the diet or age of the flies. The higher levels of LOOH in flies maintained on 10% carbohydrate did not reduce their lifespan, from which we infer that oxidative damage to only one class of biomolecules, particularly lipids, is not sufficient to influence lifespan.     

Insect walking techniques on thin stems

The attachment ability of insects on surfaces are associated not only with the micro- and nanostructure of the adhering part of an attachment device, but also with the global scale kinematics responsible for contact formation and release. In the present study, the locomotory techniques of several representatives of insects from four different orders (Orthoptera, Heteroptera, Coleoptera, and Hymenoptera), possessing different types of attachment structures, are described. The study is based on video recordings of insects walking on a flat surface and on cylindrical rods of various thickness, imitating plant stems. Attachment devices of tarsi and pretarsi were visualized using Scanning Electron Microscopy. The results show a different manner in the use of adhesive structures on substrates with various curvatures. Insects bearing attachment pads on proximal tarsomeres usually touch flat and curved substrates using all tarsomeres, whereas insects with their attachment devices on the distal tarsomeres usually walk on flat surfaces using the distal tarsomeres of the overextended tarsus. On substrates, with diameters comparable to or larger than the tarsus length, insects walk above the stem by clasping the stem with the bent tarsi. On thin stems, insects clasp the stem between their tarsi and hang under the stem. Thus, on thin and thick rods, forces applied to attachment organs act in opposite directions. There are two methods of leg positioning for walking on a rough flat substrate. In the first case, the tarsus is straightened and the rough substrate is gripped between the claws and the proximal complex of attachment devices (tarsal euplantulae, fossulae spongiosa, and terminal spurs of tibiae). In the second case the tibia does not touch the substrate; the insect is supported only by distal tarsomeres. The tarsus is in an overextended condition. On rods, with diameters comparable to or larger than the tarsus length, insects walk by clasping the stem with the bent tarsi. This posture is characteristic for the majority of insects independent of the tarsal position they normally use while walking on a plane. If the rod’s diameter is smaller than the tarsus length, walking insects usually clutch it between contralateral tarsi. Using such a posture they are supported by interlocking or by strong friction, generated by attachment devices of the proximal tarsomeres, and do not use attachment devices of the pretarsus. Contact with the substrate is reinforced due to the coordinated contralateral clutch using all supporting legs. It is concluded that the use of different types of attachment structures correlates with locomotory techniques. Handling Editor: Heikki Hokkanen     

Downstream elements of mammalian pre-mRNA polyadenylation signals: primary,secondary and higher-order structures

Primary, secondary and higher-order structures of downstream elements of mammalian pre-mRNA polyadenylation signals [poly(A) signals] are re viewed. We have carried out a detailed analysis on our database of 244 human pre-mRNA poly(A) signals in order to characterize elements in their downstream regions. We suggest that the downstream region of the mammalian pre-mRNA poly(A) signal consists of various simple elements located at different distances from each other. Thus, the downstream region is not described by any precise consensus. Searching our database, we found that ~80% of pre-mRNAs with the AAUAAA or AUUAAA core upstream elements contain simple downstream elements, consisting of U-rich and/or 2GU/U tracts, the former occurring ~2-fold more often than the latter. Approximately one-third of the pre-mRNAs analyzed here contain sequences that may form G-quadruplexes. A substantial number of these sequences are located immediately downstream of the poly(A) signal. A possible role of G-rich sequences in the polyadenylation process is discussed. A model of the secondary structure of the SV40 late pre-mRNA poly(A) signal downstream region is presented.     

Calcium/calmodulin activation of two divergent glutamate decarboxylases from tobacco

Glutamate decarboxylase (GAD, EC 4.1.1.15) catalyses the alpha-decarboxylation of glutamate to produce gamma-aminobutyrate (GABA). The nucleotide sequences of two divergent GADs (designated GAD1 and GAD3) were isolated from a Nicotiana tabacum L. cv. Samsun NN leaf cDNA library. Open reading frames indicated that GAD1 encodes a polypeptide of 496 amino acids and has greater than 99% identity with known tobacco GADs, whereas GAD3 encodes a polypeptide of 491 amino acids and has about 14% divergence from known tobacco GADs. Genomic DNA analysis suggested that there are at least four tobacco GAD genes, existing in pairs of highly identical genes. An in vitro assay at pH 7.3 revealed that activities of the recombinant proteins, after isolation from Escherichia coli and partial purification by nickel-affinity chromatography, are 57-133 times the control levels in the presence of 0.5 mM calcium and 0.2 micro M bovine calmodulin.     

Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base.

Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.     

Structural transitions in polycytidylic acid: proton buffer capacity data

The pH-dependences of proton buffer capacity of poly(C) were computed on the basis of the literature data. In these curves there were observed four peaks: two narrow and two wide ones. The first narrow peak reflects the process of cooperative formation of double helices, which is induced by protonation of the N3 atom of nucleotide bases. The first wide peak is assigned to noncooperative process of poly(C) double helices protonation at the N3 nitrogen atom. It is proposed that the second wide peak corresponds to noncooperative protonation of the neutral cytosine bases at the oxygen atom. This reaction causes cooperative dissociation of the poly(C) double helices. The second narrow peak reflects the dissociation process.     

Cardiovascular Complications Secondary to Graves’ Disease: A Prospective Study from Ukraine

Background

Graves’ disease (GD) is a common cause of hyperthyroidism resulting in development of thyrotoxic heart disease (THD).

Objectives

to assess cardiovascular disorders and health related quality of life (HRQoL) in patients with THD secondary to GD.

Patients and Methods

All patients diagnosed with THD secondary to GD between January 2011 and December 2013 were eligible for this study. Clinical assessment was performed at baseline and at the follow-up visit after the restoring of euthyroid state. HRQoL was studied with a questionnaire EQ-5D-5L.

Results

Follow-up data were available for 61 patients, but only 30 patients with THD secondary to GD were consented to participate in investigation of their HRQoL. The frequency of cardiovascular complications was significantly reduced as compared before and after the antithyroid therapy as follows: resting heart rate (122 vs. 74 bpm), blood pressure: systolic (155 vs. 123 mm Hg), diastolic (83 vs. 66 mm Hg), supraventricular premature contractions (71% vs. 7%), atrial fibrillation (72% vs. 25%), congestive heart failure (69% vs. 20%), thyrotoxic cardiomyopathy (77% vs. 26%), all p<0.01. Anti-TSH receptor antibodies were determined as independent predictor of left ventricular geometry changes, (b-coefficient = 0.04, 95%CI 0.01–0.07, p = 0.02). HRQoL was improved in all domains and self-rated health increased from 43 to 75 units by visual analogue score (p<0.001).

Conclusions

Restoring of euthyroid state in patients with GD is associated with significant elimination of cardiovascular disorders and improvement of HRQoL. To our knowledge this is the first study evaluating Ukrainian patients with THD secondary to GD with focus on HRQoL.     

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.