Impact of CT-based diagnostic imaging on management and outcome of nonfunctioning pancreatic tumors.

BACKGROUND: Sporadic malignant non-functioning pancreatic endocrine tumors (NF-PETs) are an important subset of pancreatic neoplasms. The aim of this study was to assess the impact of improved imaging on these features in a tertiary referral centre within a 20-year follow-up. PATIENTS AND METHODS: From 1988 to 2009, 51 patients were treated for sporadic malignant NF-PETs. Forty-one patients who underwent tumor resection were retrospectively attributed according to the date of the initial diagnosis, group 1: 1988-1999 vs. group 2: 2000-2009. RESULTS: Cross-sectional imaging led to positive prediction of NF-PETs in all patients. Curative resection was achieved in 76%. Synchronous metastases were present in 56% with a positive prediction of 43%. In group 1, the mean reported CT-determined tumor size was 56 vs. 54 mm in group 2 (p = 0.89). Synchronous metastases were present in 61% in group 1 vs. 57% (p = 0.99) in group 2. Metachronous metastases were recorded in 39% in group 1 vs. 43% (p = 0.84) in group 2. The mean interval from initial resection to diagnosis of metastatic disease was significantly shorter (p = 0.01) in patients from group 1 (14 vs. 61 months). Cumulative 5- and 10-year survival rates were 77% and 72% in group 1 vs. a 5-year survival rate of 66% in group 2. CONCLUSION: So far, improved CT-based imaging has no impact on earlier detection of initial synchronous metastases in sporadic malignant NF-PETs, while metachronous metastases are detected earlier.

Membrane transport: ubiquitylation in endosomal sorting.

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.

Uracil-induced down-regulation of the yeast uracil permease.

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.

Bovine serum albumin loaded poly(lactide-co-glycolide) microspheres: the influence of polymer purity on particle characteristics.

To study the influence of polymer purity on microsphere characteristics, bovine serum albumin (BSA) loaded biodegradable microspheres were prepared by spray drying using two samples of poly(lactide-co-glycolide), PLG, (50:50, mwt = 35 and 69 kDa). Polymer properties were varied by DL-lactide and glycolide addition or by ultrafiltration. While the effective drug loading was not affected by polymer purity, Tg was decreased with increasing monomer and oligomer content. The removal of these low molecular weight substances by ultrafiltration led to a narrower molecular weight distribution compared to the untreated PLG. Concerning the polymer with the higher molecular weight, microsphere morphology was also strongly affected by polymer composition. In contrast to the non-modified PLG, monomer addition yielded particles with a much smoother surface structure. Moreover, in vitro cytotoxicity of the microspheres prepared from the polymer pretreated by ultrafiltration was significantly reduced, whereas monomer addition caused a dramatic decrease of cells surviving contact with the microsphere extract. The in vivo degradation rate of the ultrafiltered microspheres was decreased and as a result, protein release at later times was slowed down. Furthermore, depending on the effective drug loading level, monomer addition resulted in a decrease in the initial protein burst. It can be concluded that the effect of low molecular weight impurities in a polymer on microsphere characteristics and on cytotoxicity cannot be ignored. Their elimination is possible by ultrafiltration.

Dielectric and thermodynamic properties of biodegradable poly(D,L-lactide-co-glycolide) and the effect on the micro-encapsulation and release of captopril.

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.

Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease.

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.

NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase.

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.

The yeast plasma membrane uracil permease is stabilized against stress induced degradation by a point mutation in a cyclin-like "destruction box".

Yeast uracil permease appears to be fairly stable in exponentially growing cells, but it undergoes rapid endocytosis followed by degradation when cells are submitted to adverse conditions, such as nutrient starvation or inhibition of protein synthesis. Uracil permease has a sequence (RIALGSLTD) that is very similar to the "destruction box" of mitotic cyclins. This box is required for the ubiquitin-dependent proteolysis of cyclins. We replaced the invariant arginine residue of the putative "destruction box" in uracil permease by an alanine. The mutation significantly protected the permease against stress-induced degradation. This result suggests that ligation to ubiquitin could be a signal for uracil permease degradation.

Endocytosis and degradation of the yeast uracil permease under adverse conditions.

Yeast uracil permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to be rather stable in growing cells, but its turnover rate (half-life of about 7 h) was much faster than that of most yeast proteins. Several adverse conditions triggered the rapid degradation of uracil permease, and so a loss of uracil uptake. Turnover was rapid when yeast cells were starved of either nitrogen, phosphate, or carbon, and as they approached the stationary growth phase. Rapid permease degradation was also promoted by the inhibition of protein synthesis. The degradation of uracil permease in response to several stresses was strikingly slower in the two mutants, end3 and end4, that are deficient in the internalization step of receptor-mediated endocytosis. Thus, internalization is the first step in the permease degradative pathway. Uracil permease is degraded in the vacuole, since pep4 mutant cells lacking vacuolar protease activities accumulated large amounts of uracil permease, which was located within the vacuole by immunofluorescence. We have yet to determine whether adverse conditions enhance permease endocytosis and subsequent degradation or divert internalized uracil permease from a recycling to a degradative pathway.

Conventional and enantioselective determination of a new blood glucose-lowering agent in biological fluids using liquid-liquid extraction and high-performance liquid chromatography.

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%.

Predictability of the covalent binding of acidic drugs in man.

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.

In vivo phosphorylation of the yeast uracil permease.

The uptake of uracil by the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. This uracil permease is a multispanning membrane protein that follows the secretory pathway to the plasma membrane. We have used in vivo pulse labeling and immunoprecipitation to show that the uracil permease is phosphorylated. Phosphoamino acid analysis indicates that the phosphorylation occurs on seryl residues. Experiments with temperature sensitive secretory mutants, blocked at successive steps of the secretory pathway, have established that the phosphorylation of the permease takes place at the plasma membrane. Under steady state conditions, Western immunoblotting showed multiple phosphorylated permease species. Their relative abundance appeared susceptible to metabolic conditions. This study is, therefore, a first step toward identifying a molecular mechanism involved in the post-translational control of a yeast transporter.

Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein.

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)

Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.

In vitro enantioselective glucuronidation of fenoprofen.

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.

Stereoselective analysis of fenoprofen and its metabolites.

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.

[Spectroscopic properties of the partial beta-agonist doxaminol (BM 10.188) and its metabolites]. / Spektroskopische Eigenschaften des partiellen beta-agonisten Doxaminol (BM 10.188) und seiner Stoffwechselprodukte.

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.

In vivo translocation of the cell wall acid phosphatase across the yeast endoplasmic reticulum membrane: are there multiple signals for the targeting process?

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall protein that follows the yeast secretory pathway. We had previously described the in vivo fate of a multicopy plasmid-encoded modified protein, lacking 15 out of 17 signal peptide amino acids. This modified protein accumulates mainly within the cell as an inactive unglycosylated form. However 30% of this precursor is translocated, glycosylated and dispatched to the cell wall. We establish, in the present report, that this phenomenon did not result from an overproduction of the plasmid encoded protein, since it was also observed in a normal single copy situation. The secretion persisted after a deletion including the single hydrophobic segment present in the N-terminus of the mature protein. The entry of both wild type and mutant APase into the ER was inhibited in sec62 mutants suggesting that the SEC62 gene product would not be implicated in signal peptide recognition.