Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S---CH₂CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP–antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP–antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP–antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.     

Morphological and molecular investigation of polymorphism and cryptic species in tanaid crustaceans: implications for tanaid systematics and biodiversity estimates

A combination of traditional taxonomic procedures and molecular techniques has provided new insight into the problems of cryptic species and sexual and ontogenetic polymorphism in the Tanaidacea. Using polymerase chain reaction and DNA markers, three cryptic species of Paratanais were identified. PCR primers were used to amplify the divergent internal transcribed spacers (ITS) of these species. Restriction digestion of the amplified rDNA generated species specific DNA banding. Male and five female stages of Paratanais maleticus sp. nov. and two other new species, P. malign us and P. perturbatius , are described. Morphological variation, both sexual and ontogenetic, was found in several generic characters of Paratanais and required the diagnosis to be modified. The identification of three undescribed cryptic species from a single microhabitat in a well studied, shallow water and easily accessible locality, demonstrate that the biodiversity of tanaid crustacean is significantly underestimated.     

Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.     

Isolation and characterization of microsatellite loci for alligator gar (Atractosteus spatula) and their variability in two other species (Lepisosteus oculatus and L. osseus) of Lepisosteidae

We report on the isolation of 17 polymorphic microsatellite loci from alligator gar (Atractosteus spatula), a large-bodied species that has experienced population declines across much of its range. These loci possessed 2-19 alleles and observed heterozygosities of 0-0.974. All loci conformed to Hardy-Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Nine and eight of these loci were found to be polymorphic in the related species Lepisosteus oculatus and L. osseus, respectively. These microsatellite loci should prove useful in conservation efforts of A. spatula through the study of population structure and hatchery broodstock management.     

Ultra‐sensitive fluorescent sensor for intracellular miRNA based on enzyme‐free signal amplification with carbon nitride nanosheet as a carrier

A novel ultra‐sensitive fluorescent sensor for monitoring microRNA (miRNA) in living cells was constructed by utilizing a hybridization chain reaction (HCR) as the signal amplification with a carbon nitride nanosheet (CNNS) as a carrier. The Cy5‐labeled hairpin DNA could be adsorbed onto the surface of CNNS, resulting in fluorescence quenching of Cy5. When treated with complementary miRNA, the fluorescence was recovered because miRNA could efficiently trigger an HCR, which led to the release of the HCR products from the CNNS. This intracellular HCR strategy can be used for ultra‐sensitive monitoring of intracellular miRNA. The main advantages of the proposed method are its simplicity, high sensitivity, high specificity and low toxicity for monitoring low‐level biomarkers.     

On the challenges of detecting whole Staphylococcus aureus cells with biosensors

Due to the increasing number of nosocomial infections and multidrug‐resistant bacterial strains, Staphylococcus aureus is now a major worldwide concern. Rapid detection and characterization of this bacterium has become an important issue for biomedical applications. Biosensors are increasingly appearing as low‐cost, easy‐to‐operate and fast alternatives for rapid detection. In this review, we will introduce the main characteristics of S. aureus and will focus on the interest of biosensors for a faster detection of whole S. aureus cells. In particular, we will review the most promising strategies in the choice of ligand for the design of selective and efficient biosensors. Their specific characteristics as well as their advantages and/or disadvantages will also be commented.