Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2′ nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2′-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2′ of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pK(a). On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pK(a) consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data

Hachimoji DNA and RNA: A genetic system with eight building blocks

Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos

New Strategies for Exploring RNA's 2′-OH Expose the Importance of Solvent during Group II Intron Catalysis

AbstractThe 2′-hydroxyl group contributes inextricably to the functional behavior of many RNA molecules, fulfilling numerous essential chemical roles. To assess how hydroxyl groups impart functional behavior to RNA, we developed a series of experimental strategies using an array of nucleoside analogs. These strategies provide the means to investigate whether a hydroxyl group influences function directly (via hydrogen bonding or metal ion coordination), indirectly (via space-filling capacity, inductive effects, and sugar conformation), or through interactions with solvent. The nucleoside analogs span a broad range of chemical diversity, such that quantitative structure activity relationships (QSAR) now become possible in the exploration of RNA biology. We employed these strategies to investigate the spliced exons reopening (SER) reaction of the group II intron. Our results suggest that the cleavage site 2′-hydroxyl may mediate an interaction with a water molecule

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated

Luminescence Sensors Applied to Water Analysis of Organic Pollutants—An Update

The development of chemical sensors for environmental analysis based on fluorescence, phosphorescence and chemiluminescence signals continues to be a dynamic topic within the sensor field. This review covers the fundamentals of this type of sensors, and an update on recent works devoted to quantifying organic pollutants in environmental waters, focusing on advances since about 2005. Among the wide variety of these contaminants, special attention has been paid polycyclic aromatic hydrocarbons, pesticides, explosives and emerging organic pollutants. The potential of coupling optical sensors with multivariate calibration methods in order to improve the selectivity is also discussed

New insights on Celtic migration in Hungary and Italy through the analysis of non-metric dental traits

The Iron Age is characterized by an extended interweaving of movements by Celts in Europe. Several waves of Celts from Western and Central Europe migrated southeast and west from the core area of the La Téne culture (between Bourgogne and Bohemia). Through the analysis of non-metric dental traits, this work aims to understand the biological relationship among Celtic groups arrived in Italy and the Carpathian Basin, as well as between local populations and Celtic newcomers. A total of 10 non-metric dental traits were analyzed to evaluate biological affinities among Celts (Sopron-Krautacker and Pilismarót-Basaharc) and Scythians-related populations from Hungary (Tápiószele), Celts from continental Europe (Switzerland and Austria), two Iron Age Etruscan-Celtic sites from northern Italy (Monterenzio Vecchio and Monte Bibele), 13 Iron Age central-southern Italic necropolises, and the northern Italian Bronze Age necropolis of Scalvinetto. Strontium isotopes were measured on individuals from the necropolis of Monte Bibele to infer their local or non-local origin. Results highlight the existence of statistically significant differences between Celts and autochthonous Italian groups. Celtic groups from Hungary and Italy (i.e., non-local individuals of Monterenzio Vecchio and Monte Bibele) share a similar biological background, supporting the historical records mentioning a common origin for Celts migrated to the eastern and southern borders of today’s Europe. The presence of a supposed Steppean ancestry both in Celts from Hungary and Celts from northern Italy corroborates the hypothesis of the existence of a westward migration of individuals and genes from the Steppe towards northern Italy during the Bronze and Iron Age, which contributed to the biological variability of pre-Celtic and later Celtic populations, respectively. Conversely, individuals from central-southern Italy show an autochthonous pre-Iron Age background. Lastly, this work supports the existence of Celtic migratory routes in northern Italy, as shown by biological and cultural admixture between Celts and Italics living together

Thyroid papillary carcinoma arising in ectopic thyroid tissue within a neck branchial cyst

BACKGROUND: Thyroid gland derives from one median anlage at the base of the tongue, and from the two fourth branchial pouches. A number of anomalies may occur during their migration. These can be in form of ectopic tissues, which are frequently found along the course of thyroglossal duct and rarely in other sites, many of these may develop same diseases as the thyroid gland. CASE PRESENTATION: A 36-years-old female presented with a 3 month history of left side neck mass. The mass disappeared following aspiration of brown colored fluid, which on cytological examination showed cells with nuclear irregularities that warranted the resection of the lesion. The histology demonstrated a thyroid papillary carcinoma arising within the branchial cyst. Thereafter, the patient underwent a total thyroidectomy with central lymph nodes dissection. Histology showed a multifocal papillary carcinoma with central lymph nodes metastases. Only four cases of primary thyroid carcinomas in neck branchial cyst have been described so far. CONCLUSION: In a lateral cystic neck mass, although rare, occurrence of ectopic thyroid tissue and presence of a papillary thyroid carcinoma should be kept in mind