Induction of mutants in Saccharomyces cerevisiae by guanidine hydrochloride II. Conditions that prevent induction

The induction of ⁻ “petite” mutants by guanidine hydrochloride (GuHCl) is inhibited in several conditions. Anaerobiosis inhibited the induction either with or without cell multiplication. Both nalidixic acid (NA) and cycloheximide (CH) inhibited the induction of mutants. On the other hand, chloramphenicol (CAP) produced a dual effect: at low concentration it stimulated, at high concentration it inhibited, the induction. The effect of these different inhibitors on the transformation of ⁺ mother cells into ⁻ by GuHCl is discussed.     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Morphometric Studies of Mitochondria in Tetrahymena pyriformis Exposed to Chloramphenicol or Ethidium Bromide*

Cultures of Tetrahymena pyriformis strain ST were exposed to 300 μg chloramphenicol/ml or 15 μg ethidium bromide/ml for 48 hr. Qualitative assessments of electron micrographs reveal that the abundance of mitochondrial cristae decreases greatly. By equating the spatial characteristics of the organism with those of a prolate spheroid, the distribution and abundance of mitochondria were quantified. Such characterizations reveal that the size of individual mitochondria decreases by 35–60% and that the number of mitochondria/cell increases ～8 fold. The observations are discussed in terms of coordinated mitochondrial and nuclear genetic activities.     

Natural products as modulators of the nuclear receptors and metabolic sensors LXR,FXR and RXR

Nuclear receptors (NRs) represent attractive targets for the treatment of metabolic syndrome-related diseases. In addition, natural products are an interesting pool of potential ligands since they have been refined under evolutionary pressure to interact with proteins or other biological targets.This review aims to briefly summarize current basic knowledge regarding the liver X (LXR) and farnesoid X receptors (FXR) that form permissive heterodimers with retinoid X receptors (RXR). Natural product-based ligands for these receptors are summarized and the potential of LXR, FXR and RXR as targets in precision medicine is discussed.     

A sensitive fluorescent nanosensor for chloramphenicol based on molecularly imprinted polymer‐capped CdTe quantum dots

A novel fluorescent nanosensor using molecularly imprinted silica nanospheres embedded CdTe quantum dots (CdTe@SiO₂@MIP) was developed for detection and quantification of chloramphenicol (CAP). The imprinted sensor was prepared by synthesis of molecularly imprinting polymer (MIP) on the hydrophilic CdTe quantum dots via reverse microemulsion method using small amounts of solvents. The resulting CdTe@SiO₂@MIP nanoparticles were characterized by fluorescence, UV–vis absorption and FT‐IR spectroscopy and transmission electron microscopy. They preserved 48% of fluorescence quantum yield of the parent quantum dots. CAP remarkably quenched the fluorescence of prepared CdTe@SiO₂@MIP, probably via electron transfer mechanism. Under the optimal conditions, the relative fluorescence intensity of CdTe@SiO₂@MIP decreased with increasing CAP by a Stern–Volmer type equation in the concentration range of 40–500 µg L^–1. The corresponding detection limit was 5.0 µg L^–1. The intra‐day and inter‐day values for the precision of the proposed method were all <4%. The developed sensor had a good selectivity and was applied to determine CAP in spiked human and bovine serum and milk samples with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations

We present an implementation of the TOXCAT membrane protein self-association assay that measures the change in apparent free energy of transmembrane helix dimerization caused by point mutations. Quantifying the reporter gene expression from cells carrying wild-type and mutant constructs shows that single point mutations that disrupt dimerization of the transmembrane domain of glycophorin A reproducibly lower the TOXCAT signal more than 100-fold. Replicate cultures can show up to threefold changes in the level of expression of the membrane bound fusion construct, and correcting for these variations improves the precision of the calculated apparent free energy change. The remarkably good agreement between our TOXCAT apparent free energy scale and free energy differences from sedimentation equilibrium studies for point mutants of the glycophorin A transmembrane domain dimer indicate that sequence changes usually affect membrane helix-helix interactions quite similarly in these two very different environments. However, the effects of point mutations at threonine 87 suggest that intermonomer polar contacts by this side-chain contribute significantly to dimer stability in membranes but not in detergents. Our findings demonstrate that a comparison of quantitative measurements of helix-helix interactions in biological membranes and genuine thermodynamic data from biophysical measurements on purified proteins can elucidate how changes in the lipidic environment modulate membrane protein stability.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.     

Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ-Ibα-Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ₂ peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ₂ complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.