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Background: The Institute of Quality Assurance and Transparency in Health Care (IQTIG) was commissioned by the Federal Joint Committee (G-BA) to include the patient perspective in the quality assurance programme for patients undergoing percutaneous cardiac intervention (PCI) and coronary angiography ("QS PCI"). This article describes both the development methodology and the survey-based quality indicators., Methods: On the basis of a systematic literature research, focus groups with patients, interviews with doctors and an expert group meeting, quality criteria were defined which were relevant to patients. These criteria were translated into PREMs and PROMs. The questionnaires were subjected to a two-phase pretest. Quality indicators were composed by aggregating the items., Results: Altogether, 12 topic areas were identified which are relevant to patients receiving percutaneous cardiac intervention or coronary angiography. Here, communication and interaction were of high importance. In addition, information before, during and after the procedure was very relevant, and, likewise, the health care professionals' interaction with patients. Also, symptoms and treatment effects were important. In accordance with the defined topics, 19 quality indicators were developed which can display quality of health care from the patients' perspective., Conclusion: The development of PREMs and PROMs expanded the quality assurance programme "QS PCI" to include significant dimensions relevant to patients, which can, therefore, provide valuable information to improve patient-centred care., (Copyright © 2023. Published by Elsevier GmbH.)

Aims: Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation., Methods and Results: General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis., Conclusion: By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

TBC1D10C is a protein previously demonstrated to bind and inhibit Ras and Calcineurin. In cardiomyocytes, also CaMKII is inhibited and all three targeted enzymes are known to promote maladaptive cardiomyocyte hypertrophy. Here, in accordance with lack of Calcineurin inhibition in vivo, we did not observe a relevant anti-hypertrophic effect despite inhibition of Ras and CaMKII. However, cardiomyocyte-specific TBC1D10C overexpressing transgenic mice exhibited enhanced longevity. Ejection fraction and exercise capacity were enhanced in transgenic mice, but shortening of isolated cardiomyocytes was not increased. This suggests longevity resulted from enhanced cardiac performance but independent of cardiomyocyte contractile force. In further search for mechanisms, a transcriptome-wide analysis revealed expressional changes in several genes pertinent to control of heart rate (HR) including Hcn4, Scn10a, Sema3a and Cacna2d2. Indeed, telemetric holter recordings demonstrated slower atrial conduction and significantly lower HR. Pharmacological reduction of HR was previously demonstrated to enhance survival in mice. Thus, in addition to inhibition of stress signaling, TBC1D10C economizes generation of cardiac output via HR reduction, enhancing exercise capacity and survival. TBC1D10C may be a new target for HR reduction and longevity.

The yeast uracil permease, Fur4p, is downregulated by uracil, which is toxic to cells with high permease activity. Uracil promotes cell surface Rsp5p-dependent ubiquitylation of the permease, signaling its endocytosis and further vacuolar degradation. We show here that uracil also triggers the direct routing of its cognate permease from the Golgi apparatus to the endosomal system for degradation, without passage via the plasma membrane. This early sorting was not observed for a variant permease with a much lower affinity for uracil, suggesting that uracil binding is the signal for the diverted pathway. The FUI1-encoded uridine permease is similarly sorted for early vacuolar degradation in cells exposed to a toxic level of uridine uptake. Membrane proteins destined for vacuolar degradation require sorting at the endosome level to the intraluminal vesicles of the multivesicular bodies. In cells with low levels of Rsp5p, Fur4p can be still diverted from the Golgi apparatus but does not reach the vacuolar lumen, being instead missorted to the vacuolar membrane. Correct luminal delivery is restored by the biosynthetic addition of a single ubiquitin, suggesting that the ubiquitylation of Fur4p serves as a specific signal for sorting to the luminal vesicles of the multivesicular bodies. A fused ubiquitin is also able to sort some Fur4p from the Golgi to the degradative pathway in the absence of added uracil but the low efficiency of this sorting indicates that ubiquitin does not itself act as a dominant signal for Golgi-to-endosome trafficking. Our results are consistent with a model in which the binding of intracellular uracil to the permease signals its sorting from the Golgi apparatus and subsequent ubiquitylation ensures its delivery to the vacuolar lumen.

We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases., (Copyright 2002 John Wiley & Sons, Ltd.)

In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor.

In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.

The mechanical and dielectric properties of three kinds of poly(lactic acid-coglycolic acid) (PLG) with different molecular weights and polydispersities fractioned by ultrafiltration were investigated by dynamic mechanical thermal analysis (DMTA) and dielectric measurement. All samples showed typical behaviour of amorphous polymer under different fields. Two relaxation processes were found, a secondary relaxation in glassy state at low temperature and a glass transition relaxation. The molecular weights and polydispersities of PLGs influenced significantly both relaxation, especially the relaxation strength and location. The strength of secondary relaxation was reduced and the glass transition shifted to a higher temperature when the molecular weight of PLG increased and the polydispersity decreased. The shift of glass transition temperature (Tg) might decrease the motion of the macromolecules and resulted in a higher moduli of rubbery PLG at the temperature of the drug system (37 degrees C) and lowered the diffusivity of the drug in polymeric matrix and then the initial burst and fast diffusional release of captopril from commercial PLG were improved.

When yeast cells growing on a poor nitrogen source are supplied with NH4+ ions, several nitrogen permeases including the general amino acid permease (Gap1p) are rapidly and completely inactivated. This report shows that inactivation by NH4+ of the Gap1 permease is accompanied by its degradation. A functional NPl1 gene product is required for both inactivation and degradation of Gap1p. Molecular analysis of the NPl1 gene showed that it is identical to RSP5. The RSP5 product is a ubiquitin-protein ligase (E3 enzyme) whose physiological function was, however, unknown. Its C-terminal region is very similar to that of other members of the E6-AP-like family of ubiquitin-protein ligases. Its N-terminal region contains a single C2 domain that may be a Ca(2+)-dependent phospholipid interaction motif, followed by several copies of a recently identified domain called WW(P). The Npi1/Rsp5 protein has a homologue both in humans and in mice, the latter being involved in brain development. Stress-induced degradation of the uracil permease (Fur4p), a process in which ubiquitin is probably involved, was also found to require a functional NPl1/RSP5 product. Chromosomal deletion of NPl1/RSP5 showed that this gene is essential for cell viability. In the viable npi1/rsp5 strain, expression of NPl1/RSP5 is reduced as a result of insertion of a Ty1 element in its 5' region. Our results show that the Npi1/Rsp5 ubiquitin-protein ligase participates in induced degradation of at least two permeases, Gap1p and Fur4p, and probably also other proteins.

Two analytical methods are described for the determination of 2-(4-tert.-butylphenoxy)-7-(4-chlorophenyl)heptanoic acid sodium salt (I) in animal models (beagle dog and rat). Method 1 is conventional reversed-phase high-performance liquid chromatography on an octadecylsilane column with an eluent of acetonitrile-0.02 M potassium buffer (pH 3) (65:35, v/v). Method 2 is used for the enantioselective determination of I. This method uses a chiral column (Chiralcel OJ) with an eluent of n-hexane-2-propanol (95:5, v/v) containing 3 ml/l trifluoracetic acid. The analytical procedure has a recovery of more than 90%; within-run precision of less than 5.1%, and between-run precision of less than 4.3%.

Although metabolism via glucuronide conjugation has generally been considered a detoxification route for carboxylic acids, the newly discovered chemical reactivity of these conjugates, leading to covalent binding with proteins, is consistent with the toxicity observed for drugs containing the carboxylic acid moiety. Here we report that degradation rates (intramolecular rearrangement and hydrolysis) for 9 drug glucuronide metabolites show an excellent correlation (r2 = 0.995) with the extents of drug covalent binding to albumin in vitro. Furthermore, this binding capacity is predictable based on chemical structure of the acid and depends on the degree of substitution at the carbon alpha to the carboxylic acid. The in vivo covalent binding in humans for these drugs is also predictable (r2 = 0.873) when the extent of adduct formation is corrected for the measured plasma glucuronide concentrations. These results suggest that the structure of a carboxylic acid drug may predict the degree to which the corresponding acyl glucuronides will form covalent adducts that probably/possibly lead to toxicity. This information could be a useful adjunct in drug design.

Stereoselective degradation of fenoprofen (FEN) glucuronides and irreversible binding of FEN enantiomers to human serum albumin via their glucuronides were studied. At different pH values, 37 degrees C, and in the absence of albumin, degradation half-lives were diastereomeric, resulting mainly from a combination of hydrolysis and acyl migration. Lower pH enhanced FEN glucuronide stability and reduced the extent of irreversible binding. The degradation rate of R-FEN glucuronide was greater than that of the S-glucuronide (S-FEN). When human serum albumin was added to the medium, stability was decreased as compared to protein-free buffer. FEN glucuronides were readily hydrolyzed to parent drug, indicating an esterase-like activity of the albumin molecule. In vitro irreversible binding was higher for R-FEN (1.22% +/- 0.36) than for S-FEN glucuronide (0.76% +/- 0.12), when a 0.1 mM concentration of each conjugate enantiomer was incubated under physiological conditions (pH 7.4, 37 degrees C). Incubation with unconjugated FEN did not lead to measurable irreversible binding. Analysis of plasma samples from a clinical study showed that enantioselective irreversible binding of FEN to plasma proteins also occurs in vivo. After administration of a single 600-mg dose of racemic FEN to six healthy volunteers, covalent binding of R- and S-FEN to plasma proteins was measured in all subjects. The percentage of S-FEN protein adduct was greater than that of its R-enantiomer adduct. Total amounts of FEN irreversibly bound to plasma protein in vivo were also very low (1.02 +/- 0.32 and 3.23 +/- 0.85 mol/mol protein x 10(-4) for R- and S-FEN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The diastereomeric glucuronic acid conjugates are major metabolites of the nonsteroidal anti-inflammatory drug fenoprofen (FEN). Glucuronidation of FEN enantiomers was investigated with liver microsomal preparations from different species (sheep, rabbit, rat and human). The formed R- and S-FEN conjugates can be separated and quantitated directly on a C18 reversed-phase HPLC column using a mixture of acetonitrile and tetrabutylammonium sulfate buffer, pH 2.5, as mobile phase. Applying this analytical procedure, it is possible to characterize enantioselective glucuronidation of FEN. For in vitro procedures, rates of glucuronide formation are substrate (FEN) and cosubstrate (UDP glucuronic acid, UDPGA) dependent with initial rates of glucuronide formation being higher for R- than for S-FEN. The R/S ratio of the formed products was independent of UDPGA (2.5-15 mmol/l) and substrate concentrations greater than or equal to 0.4 mmol/l. Enantioselective cleavage of the formed FEN conjugates by alkaline hydrolysis and hydrolytic enzymes (R greater than S-glucuronide) can be controlled during in vitro studies by pH adjustment and the addition of enzyme inhibitors.

Reversed-phase high-performance liquid chromatographic assays have been developed to quantitate simultaneously fenoprofen and its major metabolites as well as to distinguish between their R- and S- enantiomers following a single oral dose of 600 mg racemic fenoprofen to healthy volunteers. The compounds are extracted from plasma (after precipitation of plasma protein) or assayed directly in diluted urine samples employing a gradient solution on a C18 column and ultraviolet detection. Two internal standards, ketoprofen and flunoxaprofen, are used to allow measurement of very low (0.05 microgram/ml) and high (70 microgram/ml) concentrations in each sample. R- and S-fenoprofen glucuronides can be separated directly; the 4'-hydroxyfenoprofen conjugates are measured via an indirect method by comparing the concentration of 4'-hydroxyfenoprofen before and after hydrolysis. The R- and S-enantiomers of both parent and 4'-hydroxy metabolite are derivatized with L-leucinamide via an ethyl chloroformate intermediate and subsequently analyzed on a C18 column. Concentrations of metabolites found in plasma were low when compared to parent drug. The S/R ratio of fenoprofen in plasma always exceeds 1 and increases with time after dosage while the S/R ratio of its 4'-hydroxy metabolite remains almost unchanged at 1.1. R-Fenoprofen glucuronide disappears rapidly from plasma as compared to its S-antipode; a less pronounced difference is noted between R- and S-4'-hydroxyfenoprofen conjugates. Fenoprofen is almost completely excreted as its S-acyl glucuronides; the renal clearance of unchanged drug is very low.

The biotransformation of the positively inotropically active compound N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-aminoethyl)-6,11- dihydrodibenz[b,e]-oxepine, neutral fumarate, (Doxaminol, racemic mixture of diastereomeres) in dogs is examined. The metabolits M1-M7 were isolated and their chemical structures identified by 1H-NMR, 13C-NMR and mass spectroscopic methods. 2-Hydroxy-3-phenoxypropionic acid, phenoxyacetic acid, 3-(4'-hydroxy)-phenoxy-1,2-propandiol and phenylacetic acid were formed by side chain oxidation of the parent molecule. Furthermore, the following conjugates were characterized: Doxaminol-O-glucuronide, 4'-hydroxydoxaminol-O-glucuronide, and 1-hydroxy-3-phenoxy-2-propyl sulfate.

The biotransformation of the positive inotropic compound doxaminol (N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-amino-ethyl)-6,11- dihydrodibenz[b,e]oxepine, neutral fumarate; BM 10.188) was examined in bastard shepherd dogs. Metabolic products, formed by oxidative cleavage of various side chain carbon atoms of the molecule, as well as conjugated complexes with glucuronic and sulfuric acid, were isolated from urine and plasma. As main metabolites 2-hydroxy-3-phenoxy-propionic acid and phenoxyacetic acid were formed. By means of 1HNMR and 13C-NMR spectroscopy and various mass spectroscopic methods, the chemical structures of the metabolites were elucidated.

The pharmacokinetic behaviour of doxaminol (N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-amino-ethyl)-6, 11-dihydrodibenz[b,e]oxepine, neutral fumarate; BM 10.188) was examined in dogs using peroral and intravenous application of the 14C-labelled drug. The maximum plasma concentration was reached 1 h after application, indicating a relatively quick absorption of doxaminol. Decrease of total radioactivity after intravenous and peroral application is characterized by two phases, the elimination half-lives being 1.33 and 1.55 h, respectively, and 24.05 and 21.05 h, respectively. The biological availability of doxaminol was ca. 60%. The plasma levels of the unchanged drug showed that doxaminol was very rapidly eliminated and metabolized. Within the examined period of 96 h, the elimination of doxaminol and its metabolites via urine and faeces amounted to 76.5% after intravenous application, and 44.1% of the applied dose after peroral application. The major amount of radioactivity is eliminated via faeces (61.5% and 31.2% of dose, respectively) while the elimination through urine is found to be 15.0 and 12.9% of the dose, respectively.

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.

5-Aminolevulinate synthase from yeast mitochondria has been purified to homogeneity for the first time. By using affinity chromatography on agarose-hexane-CoA, gel filtration and DEAE-Sepharose chromatography, the enzyme was purified about 7000-fold with an overall yield of 40%. The specific activity of the final preparation was 39000 nmol of 5-aminolevulinate h-1 mg-1 of protein at 30 degrees C. As judged by gel filtration, polyacrylamide gradient gel and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the enzyme appeared to be composed of two identical subunits of a relative molecular mass of 53000. Electrophoresis of sodium-dodecyl-sulfate-solubilized yeast homogenate followed by immune replica analysis showed that the value of 53000 is the Mr of a non-degraded form. The purified enzyme had an isoelectric point of 5.3 and a pH optimum of 7.4. Pyridoxal 5'-phosphate has been shown to be an essential cofactor. The enzyme activity was sensitive to thiol blocking reagents. Hemin, but not heme, inhibited the activity of the purified enzyme.