Ph. Eur. testing for histamine and depressor substances using guinea-pigs and cats: the end of an era. Strategy for removal of animal tests for histamine and depressor substances and their vestiges from the Ph. Eur.

For more than 50 years, assays have been used for testing pharmaceutical product safety due to their assumed ability to broadly detect potential unidentified contaminants. As part of these in vivo tests, the animal tests for depressor substances and histamine have been described in the European Pharmacopoeia since its first edition in 1977. Both tests measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively.

Informal investigation on the added value of a potential certification system for the qualification of raw materials for the production of ATMPs.

The manufacture of advanced therapy medicinal products (ATMPs) is critically impacted by the quality of the raw materials (RMs) used. Following the need expressed by stakeholders to establish a certification scheme for biological RMs used in the manufacture of ATMPs, the European Pharmacopoeia (Ph. Eur.

European Pharmacopoeia's Approach to Cell and Gene Therapy: Focus on How Gene Therapy Texts Are Evolving.

The European Pharmacopoeia (Ph. Eur.) constitutes single recognised common standard for the quality control of medicines in Europe, which is also applied in many countries worldwide.

Replacement, Reduction, Refinement - Animal welfare progress in European Pharmacopoeia monographs: activities of the European Pharmacopoeia Commission from 2007 to 2017.

Since the opening for signature of the European Convention for the Protection of Animals Used for Experimental and Other Scientific Purposes in 1986, the European Pharmacopoeia Commission and its experts have carried out a programme of work committed to Replacing, Reducing and Refining (3Rs) the use of animals for test purposes. While updates on achievements in the field of the 3Rs are regularly provided, this article summarises the activities of the Ph. Eur.

Structures and Activities of the Elongator Complex and Its Cofactors.

Elongator is a highly conserved eukaryotic protein complex consisting of two sets of six Elp proteins, while homologues of its catalytic subunit Elp3 are found in all the kingdoms of life. Although it was originally described as a transcription elongation factor, cumulating evidence suggests that its primary function is catalyzing tRNA modifications. In humans, defects in Elongator subunits are associated with neurological disorders and cancer.

Architecture of the yeast Elongator complex.

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive.

Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi.

During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs.

Cation Diffusion Facilitator family: Structure and function.

The Cation Diffusion Facilitators (CDFs) form a family of membrane-bound proteins capable of transporting zinc and other heavy metal ions. Involved in metal tolerance/resistance by efflux of ions, CDF proteins share a two-modular architecture consisting of a transmembrane domain (TMD) and C-terminal domain (CTD) that protrudes into the cytoplasm. Discovery of a Zn²⁺ and Cd²⁺ CDF transporter from a marine bacterium Maricaulis maris that does not possess the CTD questions current perceptions regarding this family of proteins.

Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification.

Unlabelled: Modification of wobble uridines of many eukaryotic tRNAs requires the Elongator complex, a highly conserved six-subunit eukaryotic protein assembly, as well as the Killer toxin-insensitive (Kti) proteins 11-14. Kti11 was additionally shown to be implicated in the biosynthesis of diphthamide, a post-translationally modified histidine of translation elongation factor 2. Recent data indicate that iron-bearing Kti11 functions as an electron donor to the [4Fe-4S] cluster of radical S-Adenosylmethionine enzymes, triggering the subsequent radical reaction.

Functional dissection of the multi-domain di-heme cytochrome c(550) from Thermus thermophilus.

In bacteria, oxidation of sulfite to sulfate, the most common strategy for sulfite detoxification, is mainly accomplished by the molybdenum-containing sulfite:acceptor oxidoreductases (SORs). Bacterial SORs are very diverse proteins; they can exist as monomers or homodimers of their core subunit, as well as heterodimers with an additional cytochrome c subunit. We have previously described the homodimeric SOR from Thermus thermophilus HB8 (SOR(TTHB8)), identified its physiological electron acceptor, cytochrome c(550), and demonstrated the key role of the latter in coupling sulfite oxidation to aerobic respiration.

Atypical features of Thermus thermophilus succinate:quinone reductase.

The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form.

Maricaulis maris cation diffusion facilitator: achieving homogeneity through a mixed-micelle approach.

One of the most common problems encountered during isolation and purification of homogenous membrane proteins is their aggregation which in turn makes the obtained material unsuitable for structural and functional studies. As various detergents can have a different impact on the protein stability and solubility, introducing the protein to different detergent micelles can result in the removal of such aggregation. Here we describe a method for retrieving homogenous samples of a putative member of the cation diffusion facilitator family from the marine bacterium Maricaulis maris (MmCDF3).

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy.

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.

Development of real-time PCR assays for detection and quantification of Bacillus cereus group species: differentiation of B. weihenstephanensis and rhizoid B. pseudomycoides isolates from milk.

Quantitative real-time PCR (qRT-PCR) offers an alternative method for the detection of bacterial contamination in food. This method provides the quantitation and determination of the number of gene copies. In our study, we established an RT-PCR assay using the LightCycler system to detect and quantify the Bacillus cereus group species, which includes B.

Biochemical and biophysical characterization of succinate: quinone reductase from Thermus thermophilus.

Enzymes serving as respiratory complex II belong to the succinate:quinone oxidoreductases superfamily that comprises succinate:quinone reductases (SQRs) and quinol:fumarate reductases. The SQR from the extreme thermophile Thermus thermophilus has been isolated, identified and purified to homogeneity. It consists of four polypeptides with apparent molecular masses of 64, 27, 14 and 15kDa, corresponding to SdhA (flavoprotein), SdhB (iron-sulfur protein), SdhC and SdhD (membrane anchor proteins), respectively.