Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

High irradiance improves ammonium tolerance in wheat plants by increasing N assimilation

Ammonium is a paradoxical nutrient ion. Despite being a common intermediate in plant metabolism whose oxidation state eliminates the need for its reduction in the plant cell, as occurs with nitrate, it can also result in toxicity symptoms. Several authors have reported that carbon enrichment in the root zone enhances the synthesis of carbon skeletons and, accordingly, increases the capacity for ammonium assimilation. In this work, we examined the hypothesis that increasing the photosynthetic photon flux density is a way to increase plant ammonium tolerance. Wheat plants were grown in a hydroponic system with two different N sources (10 mM nitrate or 10 mM ammonium) and with two different light intensity conditions (300 μmol photon m⁻² s⁻¹ and 700 μmol photon m⁻² s⁻¹). The results show that, with respect to biomass yield, photosynthetic rate, shoot:root ratio and the root N isotopic signature, wheat behaves as a sensitive species to ammonium nutrition at the low light intensity, while at the high intensity, its tolerance is improved. This improvement is a consequence of a higher ammonium assimilation rate, as reflected by the higher amounts of amino acids and protein accumulated mainly in the roots, which was supported by higher tricarboxylic acid cycle activity. Glutamate dehydrogenase was a key root enzyme involved in the tolerance to ammonium, while glutamine synthetase activity was low and might not be enough for its assimilation.     

Brain edema in acute liver failure and chronic liver disease: Similarities and differences

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that typically develops as a result of acute liver failure or chronic liver disease. Brain edema is a common feature associated with HE. In acute liver failure, brain edema contributes to an increase in intracranial pressure, which can fatally lead to brain stem herniation. In chronic liver disease, intracranial hypertension is rarely observed, even though brain edema may be present. This discrepancy in the development of intracranial hypertension in acute liver failure versus chronic liver disease suggests that brain edema plays a different role in relation to the onset of HE. Furthermore, the pathophysiological mechanisms involved in the development of brain edema in acute liver failure and chronic liver disease are dissimilar. This review explores the types of brain edema, the cells, and pathogenic factors involved in its development, while emphasizing the differences in acute liver failure versus chronic liver disease. The implications of brain edema developing as a neuropathological consequence of HE, or as a cause of HE, are also discussed.     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents

To understand the photosynthetic basis in a single seed descent line 10 (SSDL10) of wheat contained high ATP in leaves, the chloroplast proteome was compared to SSDL10 and its parents using a combination of 2-DE and MALDI-TOF MS and MS/MS. More than 300 protein spots could be reproducibly detected in the 2D gel. 18 spots were differentially expressed between SSDL10 and the parents, 16 of which were identified by MS with the localization in chloroplasts. These proteins are grouped into diverse functional categories, including Calvin cycle and electron transport in photosynthesis, redox homeostasis, metabolism, and regulation. In addition to Rubisco large subunit, the content of photosynthetic electron transfers such as chlorophyll a-b binding protein, ATP synthase δ subunit, ferredoxin-NADP⁺ oxidoreductase (FNR) was higher in SSDL10 than in its parents. Furthermore, cyclic electron transfer around photosystem I (CET) was faster in SSDL10 than in the parents. Analysis of NADPH-NBT oxidoreductase activity combined with immuno-detection further revealed that, the activity of two high molecular mass protein complexes containing FNR probably involved, the CET appeared higher in SSDL10 than in the parents. The possible mechanism for the regulative role of CET in photosynthesis in SSDL10 is discussed.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome type A is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.     

Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity

It was hypothesized that residues Val44 and Val45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest that mutation at Val45 has more impact on the structure and stability of hGS than does mutation at Val44. Experimentally, enzymes with mutations at the 44 and or 45 positions of hGS were prepared, purified and assayed for initial activity. Val45 position mutations (either to alanine or tryptophan) have a greater impact on enzyme activity than do mutations at Val44. Differential scanning calorimetry experiments reveal a loss of stability in all mutant enzymes, with V45 mutations being less stable than the corresponding Val44 mutations. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases when tryptophan is introduced at either of these positions. Hill coefficients trend towards less negative cooperativity with the exception of V45W mutant hGS. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme, that their mutation impacts the allosteric pathway more than it does the active site of hGS, and that these residues (and by extension the dimer interface in which they are located) are integral to the stability of human glutathione synthetase.     

The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.