Secondary Quality Control Research Articles

An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review)

Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments.

Chromatographic fingerprint analysis of Cephalotaxus sinensis from various sources by high-performance liquid chromatography–diodearray detection–electrospray ionization-tandem mass spectrometry

Selective and efficient analytical methods are required not only for quality assurance but also for authentication of Chinese herbal medicine. A simple, rapid and valid fingerprint method has been first carried out for the quality control of Cephalotaxus sinensis by using high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) and electrospray ionisation-tandem mass spectrometry (ESI-MS). The characteristic analytical fingerprints of this plant extract showed 18 common peaks, and out of these, 10 compounds involving 2 new compounds were identified by comparing the retention time, UV and ESI-MS/MS spectrum of each standard with those of each peak separated by on-line HPLC–DAD–MS/MS. Moreover, the effects of collecting locations, harvesting time, storage time, drying methods, and medicinal portions on herbal chromatographic fingerprints were examined by similarity analysis and principal component analysis (PCA) along with markers. Using the reference fingerprint along with markers, the best harvesting time, cultivation location and medicinal part were determined. The results obtained suggest that the chromatographic fingerprint combining similarity evaluation and PCA along with markers or pharmacologically active constituents can efficiently identify raw herb of Cephalotaxus sinensis from different sources, which provide helpful clues to the study of plant's secondary metabolites and benefit quality control.

Missense Mutations of Dual Oxidase 2 (DUOX2) Implicated in Congenital Hypothyroidism Have Impaired Trafficking in Cells Reconstituted with DUOX2 Maturation Factor

Dual oxidase 2 (DUOX2), a reduced NAD phosphate:O2 oxidoreductase flavoprotein, is a component of the thyrocyte H2O2 generator required for hormone synthesis at the apical plasma membrane. We recently identified a specific DUOX2 maturation factor (DUOXA2) that is necessary and sufficient for expression of functional DUOX2 in mammalian cell lines. We have now used a DUOXA2 reconstituted system to provide the first characterization of natural DUOX2 missense variants (Q36H, R376W, D506N) at the molecular level, analyzing their impact on H2O2 generation, trafficking, stability, folding, and DUOXA2 interaction. The Q36H and R376W mutations completely prevent routing of DUOX2 to the cell surface. The mutant proteins are predominantly present as core N-glycosylated, thiol-reduced folding intermediates, which are retained by the quality control system within the endoplasmic reticulum (ER) as indicated by increased complexation with the lectin calnexin. D506N displays a partial deficiency phenotype with reduced surface expression of a mutant protein with normal intrinsic activity in generating H2O2. D506N N-glycan moieties are not subject to normal modification in the Golgi apparatus, suggesting that nonnative protein can escape the quality control in the ER. Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of DUOX2, but is not facilitated by DUOXA2. Rather, DUOXA2 allows rapid ER exit of folded DUOX2 or enhanced degradation of mutant DUOX2 proteins not competent for ER exit. DUOXA2 may thus be part of a secondary quality control system specific for DUOX2.

A preliminary study of plant aroma profile characteristics by a combination sampling method coupled with GC–MS

Plant aroma profile characteristic is an important bio-information. In this study, a sampling method in combination with headspace solid phase microextraction (HSSPME), simultaneous distillation extraction (SDE) and steam distillation (SD), followed by gas chromatography–mass spectrometry (GC–MS) detection, was used to study the aroma profile characteristics of the fresh and stale samples of common tomato, cherry tomato, durian and longan, and of the different samples of Chinese mango and Allium varieties. The typical aroma volatiles of these samples were isolated and identified according to the different degrees of certainty. The different samples showed different aroma profile characteristics when principal component analysis (PCA) was conducted to analyze the data of the aroma profile chromatograms. Then the crucial aroma volatiles contributing greatly to the clustering differences of the aroma profile characteristics of the fresh and stale common tomato, cherry tomato, durian and longan were identified by common model strategy. These compounds are potential bio-markers of plant metabolism, but further study is needed. Continuous investigations for the aroma profile characteristics of common tomato during storage and chive during growth were conducted to distill the potential bio-information from the plant metabolism processes. The saturated hexanal of common tomato increased during storage, whereas the unsaturated hexenal reduced. The accumulating trends of volatile sulfides were observed during chive growth. The preliminary results related with the corresponding bio-information could provide helpful clues to the study of plant's secondary metabolism process and benefit quality control.

Presenilin-1 Maintains a Nine-Transmembrane Topology throughout the Secretory Pathway

Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active gamma-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.

The Use of Embolic Signal Detection in Multicenter Trials to Evaluate Antiplatelet Efficacy

The CARESS (Clopidogrel and Aspirin for Reduction of Emboli in Symptomatic carotid Stenosis) trial proved the effectiveness of the combination of clopidogrel and aspirin compared with aspirin alone in reducing presence and number of microembolic signals (MES) in patients with recently symptomatic carotid stenosis. The present study aimed at installing primary and secondary quality control measures in CARESS because MES evaluation relies on subjective judgment by human experts. As primary quality control, centers participating in CARESS evaluated a reference digital audio tape (DAT) before the study containing both MES and artifacts. Interobserver agreement of classifying signals as MES was expressed as proportions of specific agreement of positive ratings (ps+/-values). For all DATs included in CARESS (n=300), online number of MES and off-line number of MES read by the central reader were compared using correlation coefficients. As secondary control, a sample of 16 of 300 DATs was cross-validated by another independent reader (post-trial validator). For the reference tape, the cumulative ps+/-value was 0.894 based on 12 of 14 observers. Two observers with very different results improved after a training procedure. Agreement between post-trial validator and central reader was ps+=0.805, indicating very good agreement. Correlation between online evaluation and off-line evaluation of DATs was very good overall (cumulative rho=0.84; P<0.001). Multicenter studies using MES as outcome parameter are feasible. However, primary and secondary quality control procedures are important.

Major histocompatibility class I folding, assembly, and degradation: A paradigm for two-stage quality control in the endoplasmic reticulum

Protein folding in living cells is a complex process involving many interdependent factors. The primary site for folding of nascent proteins destined for secretion is the endoplasmic reticulum (ER). Several disease states, including cystic fibrosis, are brought about because of irregularities in protein folding. Under normal cellular conditions, "quality control" mechanisms ensure that only correctly folded proteins are exported from the ER, with incorrectly folded or incompletely assembled proteins being degraded. Quality control mechanisms can be divided into two broad processes: (1) Primary quality control involves general mechanisms that are not specific for individual proteins; these monitor the fidelity of nascent protein folding in the ER and mediate the destruction of incompletely folded proteins. (2) Partially folded or assembled proteins may be subject to secondary quality control mechanisms that are protein- or protein-family-specific. Here we use the folding and assembly of major histocompatibility complex (MHC) class I as an example to illustrate the processes of quality control in the ER. MHC class I, a trimeric complex assembled in the ER of virally infected or malignant cells, presents antigenic peptide to cytotoxic T lymphocytes; this mediates cell killing and thereby prevents the spread of infection or malignancy. The folding and assembly of MHC class I is subjected to both primary and secondary quality control mechanisms that lead either to correct folding, assembly, and secretion or to degradation via a proteasome-associated mechanism.