Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

Der einfluß von faseroberflächen auf die matrixhärtung bei faserverbundwerkstoffen

The influence of extraction of reinforced fibres on surface properties and the curing reaction of epoxy resin matrix in composites were investigated. With increasing fibre surface polarity, as the result of the extraction, the resin wettability was improved and the reaction rate of curing of the epoxy resin matrix increased. The wettability and the reaction rate oppositely decreased with decreasing fibre surface polarity.     

Molecular Weight Characterization and Gelling Properties of Acid-Modified Maize Starches

The reduction in the molecular weight distribution during acid hydrolysis of ordinary and waxy maize starch occured in two stages: first amylopectin degraded to intermediate high molecular weight polysac-charides, secondly, these were further hydrolyzed. The amylose moiety of ordinary and high-amylose starch was not affected very much by the hydrolysis conditions used (0.1–1 M HCI, 40°C, 0.3–4 h), but the gelling properties of starch weakened. The amylopectin degradation products, probably branched dextrins, were shown to hinder the gel formation of amylose in the starch pastes.     

Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient’s plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.     

Photochemie von Acylaziden. VIII [1]. Reagieren Acylnitrene wie 1,3-Dipole??

Photochemistry of Acylazides. VIII. Do Acylnitrenes React like 1,3-Dipoles? The formation of three- ore five-membered heterocyclic rings by the reaction of acylnitrenes with olefins depends on the electron density at the double bond. The generally expected formation of aziridines by a cheletropic reaction was observed by photolysis of aroylazides 1 in the presence of 2,5-dihydrofuran 2 . But with enolethers 3 and 4 oxazolines were directly formed. This [3 + 2] cycloaddition is regiospecific. The cycloaddition is modestly stereoselective by steric hindrance within the cyclic enolether 3b . Very small de-values were found with chiral substituents in the acylazide 13 . The azide decomposition was also achieved by photoinduced electron transfer. The same cycloaddition products as obtained by direct photolysis of the azides were obtained via radical anions of the acylazide. Using Michler's ketone as electron donor in the triplet state the formation of isocyanate which diminishes the yield of cycloadducts can be avoided.     

Chemical Stability of α‐Tocopherol in Colloidal Lipid Particles with Various Morphologies

Colloidal lipid particles (CLPs) are promising encapsulation systems for lipophilic bioactives, such as oil‐soluble antioxidants that are applied in food and pharmaceutical formulations. Currently, there is no clear consensus regarding the relation between particle structure and the chemical stability of such bioactives. Using α‐tocopherol as a model antioxidant, it is shown that emulsifier type (Tween 20 or 40, or sodium caseinate) and lipid composition (tripalmitin, tricaprylin, or combinations thereof) modulated particle morphology and antioxidant stability. The emulsifier affects particle shape, with the polysorbates facilitating tripalmitin crystallization into highly ordered lath‐like particles, and sodium caseinate resulting in less ordered spherical particles. The fastest degradation of α‐tocopherol is observed in tripalmitin‐based CLPs, which may be attributed to its expulsion to the particle surface induced by lipid crystallization. This effect is stronger in CLPs stabilized by Tween 40, which may act as a template for crystallization. This work not only shows how the architecture of CLPs can be controlled through the type of lipid and emulsifier used, but also gives evidence that lipid crystallization does not necessarily protect entrapped lipophilic bioactives, which is an important clue for encapsulation system design. Practical Applications: Interest in enriching food and pharmaceutical products with lipophilic bioactives such as antioxidants through encapsulation in lipid particles is growing rapidly. This research suggests that for efficient encapsulation, the particle architecture plays an important role; to tailor this, the contribution of both the lipid carrier and the emulsifier needs to be considered.     

Isolation of adipophilin and butyrophilin from bovine milk and characterization of a cDNA encoding adipophilin

The milk fat globule membrane-associated proteins adipophilin (alias adipocyte differentiation-related protein) and butyrophilin were purified from bovine milk by reverse-phase chromatography. The nucleotide sequence of bovine adipophilin was obtained via peptide mapping and sequencing of a mammary gland cDNA clone, which comprises 1841 nucleotides and has an open reading frame of 450 amino acids. By peptide mapping, 19% of the amino acid sequence was confirmed. The obtained amino acid sequence has 87 and 80% identical residues with human and mouse adipophilin, respectively. Alignment with the proteins perilipin and TIP47 revealed two highly conserved segments, which may assemble into amphipathic alpha-helices.     

Prevalence, distribution, and diversity of Listeria monocytogenes in retail environments, focusing on small establishments and establishments with a history of failed inspections

Despite growing concerns about cross-contamination of ready-to-eat foods with Listeria monocytogenes, our knowledge about the ecology and transmission of L. monocytogenes in retail establishments has remained limited. We conducted a cross-sectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments that were hypothesized to present an increased risk for environmental L. monocytogenes contamination (i.e., small establishments and establishments with a history of failed New York State Agriculture and Markets inspections). Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. L. monocytogenes subtype diversity differed between sites, with lineage I isolates significantly associated with nonfood contact surfaces and lineage II isolates significantly associated with food contact surfaces. Isolates belonging to the same ribotype were often found dispersed across multiple sites within an operation.     

Extraction of heavy metals from soils using biodegradable chelating agents

Metal pollution of soils is widespread across the globe, and the clean up of these soils is a difficulttask. One possible remediation technique is ex-situ soil washing using chelating agents. Ethylenediaminetetraacetic acid (EDTA) is a very effective chelating agent for this purpose but has the disadvantage that it is quite persistent in the environment due to its low biodegradability. The aim of our work was to investigate the biodegradable chelating agents [S,S]-ethylenediaminedisuccinic acid (EDDS), iminodisuccinic acid (IDSA), methylglycine diacetic acid (MGDA), and nitrilotriacetic acid (NTA) as potential alternatives and compare them with EDTA for effectiveness. Kinetic experiments showed for all metals and soils that 24 h was the optimum extraction time. Longer times only gave minor additional benefits for heavy metal extraction but an unwanted increase in iron mobilization. For Cu at pH 7, the order of the extraction efficiency for equimolar ratios of chelating agent to metal was EDDS > NTA> IDSA > MGDA > EDTA and for Zn it was NTA > EDDS > EDTA >MGDA > IDSA. The comparatively low efficiency of EDTA resulted from competition between the heavy metals and co-extracted Ca. For Pb the order of extraction was EDTA > NTA >EDDS due to the much stronger complexation of Pb by EDTA compared to EDDS. At higher concentration of complexing agent, less difference between the agents was found and less pH dependence. There was an increase in heavy metal extraction with decreasing pH, but this was offset by an increase in Ca and Fe extraction. In sequential extractions EDDS extracted metals almost exclusively from the exchangeable, mobile, and Mn-oxide fractions. We conclude that the extraction with EDDS at pH 7 showed the best compromise between extraction efficiency for Cu, Zn, and Pb and loss of Ca and Fe from the soil.     

Identification of chemical markers for the sensory shelf-life of saveloy

The aroma composition, the microbial composition and the sensory profile were measured in sliced saveloy samples packed in modified atmosphere (MA). The main objective was to identify aroma compounds with potential as chemical markers to identify the sensory changes of saveloy. The 60 aroma compounds isolated from the saveloy samples by dynamic headspace extraction and measured by Gas Chromatography Mass Spectrometry (GC-MS) were used to model the sensory attributes sour&old odour and meaty odour using partial least squares regression (PLS). 2- and 3-methylbutanal, 2- and 3-methylbutanol, acetoin and diacetyl were found to have the highest impact on both sour&old odour and meaty odour of the samples. The results show that these four aroma compounds have high potential as chemical markers for the sensory shelf-life of sliced and MA-packed saveloy.