Purification and characterization of an endopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The K_M value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co²⁺ and Mg²⁺, no effect was observed with Ca²⁺. After inhibition with EDTA, enzyme activity could be restored fully by Co²⁺. Activity was inhibited by Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. The N-terminal sequence of the endopeptidase was determined as: H₂N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H.     

Mediator-Related Symptoms and Anaphylaxis in Children with Mastocytosis

Mastocytosis is characterized by the pathological accumulation of mast cells (MC) in various organs. In these patients, MC may degranulate and thereby contribute to clinical symptoms, especially when a concomitant allergy is present. However, MC activation can not only be induced by high-affinity receptors for IgE, but also by anaphylatoxins, neuropeptides, IgG immune complexes, complement-components, drugs, products of bacteria or parasites, as well as physical factors such as heat, cold, vibration, stress, sun, or physical effort. Symptoms due to mediators released by activated MC may develop in adults suffering from systemic mastocytosis, but also evolve in children who usually have cutaneous mastocytosis (CM). Clinically, CM is otherwise characterized by typical brown, maculopapular skin lesions or mastocytoma associated with a positive Darier’s sign. Pruritus and flushing are common and blistering may also be recorded, especially in diffuse CM (DCM). Pediatric patients with mastocytosis may also have gastrointestinal, respiratory, and neurologic complaints. Although anaphylaxis is not a typical finding, pediatric patients with massive skin involvement and high tryptase levels have a relatively high risk to develop anaphylaxis. This paper reviews MC mediator-related symptoms and anaphylaxis in children with mastocytosis, with special emphasis on risk factors, triggers, and management.     

An empirical study of control logic specifications for programmable logic controllers

This paper presents an empirical study of control logic specifications used to document industrial control logic code in manufacturing applications. More than one hundred input/output related property specifications from ten different reusable function blocks were investigated. The main purpose of the study was to provide understanding of how the specifications are expressed by industrial practitioners, in order to develop new tools and methods for specifying control logic software, as well as for evaluating existing ones. In this paper, the studied specifications are used to evaluate linear temporal logic in general and the specification language ST-LTL, tailored for functions blocks, in particular. The study shows that most specifications are expressed as implications, that should always be fulfilled, between input and output conditions. Many of these implications are complex since the input and output conditions may be mixed and involve sequences, timer issues and non-boolean variables. Using ST-LTL it was possible to represent all implications of this study. The few non-implication specifications could be specified in ST-LTL as well after being altered to suit the specification language. The paper demonstrates some advantages of ST-LTL compared to standard linear temporal logic and discusses possible improvements such as support for automatic rewrite of complex specifications.     

A simple method to calculate the accessible volume of protein-bound ligands: application for ligand selectivity

We here present a method for estimating the accessible volume surrounding each atom of a ligand when bound to a receptor. The accessible volumes are calculated as the volume of a sphere with fixed radius, centered on each ligand atom, not simultaneously occupied by the volume of the receptor. The method is capable of describing the packing of the ligand in the binding pocket. Moreover, where structural models of several receptors in complex with the same ligand are available, a comparative study discriminating on accessible volumes can be performed. For these cases, a relatively large accessible volume in a particular receptor might indicate that this receptor has a unique cavity that might be exploited to develop a selective ligand analog. The ligand atoms showing variation in surrounding volume accessibility when bound to different receptors constitute attractive anchor points where one might want to attach substituents that modify the selectivity of the ligand. We have applied the described method to two different enzyme-ligand systems that bind tetrahydrobiopterin, i.e. the aromatic amino acid hydroxylases and nitric oxygen synthases. Our results yield new insights into the specificity of cofactor binding to these protein families.     

Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain’s metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain’s complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.     

Quality of comminuted sausages formulated from mechanically deboned poultry meat

Comminuted sausages formulated with mechanically deboned poultry meat-MDPM (turkey or chicken, frame or neck) treated in different ways before production (vacuum packed MDPM or air packed skeletons deboned at production) and stored frozen for 6 or 18 weeks have been studied using a full-factorial design and chemical, physical and sensory analysis. MDPM was obtained from a Beehive separator. Comminuted sausages with MDPM from turkey frames, stored at -25?°C as skeletons, packed in air for 18 weeks developed marked rancid odour and flavour which could also be detected by thiobarbituric acid reactive substances (TBARS) and the concentration of volatile compounds. Vacuum packed mechanically deboned meat irrespective of species and carcass part could be stored for up to 18 weeks and used in formulation of sausages without any serious changes in their sensory profiles. Instrumental colour differences between sausages were mainly due to species. Turkey sausages were darker, redder and less yellow than chicken sausages. Moisture and fat contents were influenced by carcass part and to a lesser extent depended on species and storage time. Principal least squares regression (PLS2) showed close relationships between instrumental parameters and sensory attributes. TBARS, hexanal and volatile compounds were highly correlated with rancid flavour while redness (a(?)) was strongly related to sensory colour hue and colour strength.     

Trans-situated use of integrated information systems

Practice-based perspectives have established the situated nature of how technology is appropriated, enacted, and improvised in organisations. Empirical studies demonstrate how the same technology produces different results in different contexts of use. However, practice-based research has, to date, less to offer in terms of accounting for the relationship between instances of situated use (i.e., work practices) that are separated in space and/or time. The term trans-situated use is intended to highlight this blind spot. We focus on one type of relationship, viz., significant degrees of similarities between technologically mediated, geographically dispersed work practices. This degree of similarity is achieved through a process of commensurability consisting of (i) standardisation (addressing interdependencies between multiple instances of the ‘same’ work practice at geographically dispersed sites); and (ii) heterogeneity (addressing the entanglement of one work practice with apparently unrelated work practices and modules). Empirically, we report on a longitudinal, interpretative case study (1998–2004) of a company strategically targeting an integrated information system as a principal vehicle to establish similar services globally.     

Electrical characterization of individual metal‐coated polymer spheres used in isotropic conductive adhesives

Isotropic conductive adhesives (ICAs) filled with metal‐coated polymer spheres (MPS) have been proposed to improve the mechanical reliability compared to conventional ICAs filled with silver flakes. The electrical properties of MPS play an important role in the electrical performance of macroscopic MPS‐based ICAs. This article deals with the electrical characterization of individual MPS using a nanoindentation‐based flat punch method, in which the resistance and the deformation of single MPS were monitored simultaneously. Four groups of silver‐coated polymer spheres (AgPS) with identical polymer cores but different silver coating thicknesses were tested. The resistance of AgPS decreases gradually with increasing deformation degree of particles, and increases when the deformation of particles is reduced. In addition, the resistance of individual AgPS is dependent on the physical properties of the silver coating, such as thickness, uniformity, and porosity. The thicker the silver coating is, the lower and more stable the resistance of AgPS is. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43764.