Encapsulation of bacterial cells in electrospun microtubes

Encapsulation of whole microbial cells in microtubes for use in bioremediation of pollutants in water systems was the main focus of this investigation. Coelectrospinning of a core polymeric solution with bacterial cells and a shell polymer solution using a spinneret with two coaxial capillaries resulted in microtubes with porous walls. The ability of the microtube's structure to support cell attachment and maintain enzymatic activity and proliferation of the encapsulated microbial cells was examined. The results obtained show that the encapsulated cells maintain some of their phosphatase, β-galactosidase and denirification activity and are able to respond to conditions that induce these activities. This study demonstrates electrospun microtubes are a suitable platform for the immobilization of intact microbial cells.     

漆酶催化苯甲醇氧化制备苯甲醛

以自制的高活性漆酶为催化剂,考察漆酶催化苯甲醇制备苯甲醛的工艺条件(底物浓度、介质体系、溶剂体系、氢受体、酶的用量、通氧方式等)对氧化反应的影响。结果发现:优化反应条件为以2,2,6,6-四甲基哌啶-1-氧基(TEMPO)为介质体系且TEMPO与苯甲醇的摩尔比为1∶4、60 mmol/L的丙酮为氢受体、漆酶比酶活80 U/mL、60mmol/L的苯甲醇,反应体系通O20.5 h后密闭反应36 h,苯甲醛的产率达98%。     

Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens

Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon and energy source because it harbours the enzyme benzaldehyde lyase that cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofactor. In the reverse reaction, this lyase catalyses the carboligation of two aldehydes with high substrate and stereospecificity. The enzyme structure was determined by X-ray diffraction at 2.6 A resolution. A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes. The ThDP cofactors of these enzymes are fixed at their two ends in separate domains, suspending a comparatively mobile thiazolium ring between them. While the residues binding the two ends of ThDP are well conserved, the lining of the active centre pocket around the thiazolium moiety varies greatly within the group. Accounting for the known reaction chemistry, the natural substrate R-benzoin was modelled unambiguously into the active centre of the reported benzaldehyde lyase. Due to its substrate spectrum and stereospecificity, the enzyme extends the synthetic potential for carboligations appreciably.     

3-Hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis of benzoic acid and salicylic acid but benzaldehyde is not

Stable-isotope-labelled (²H₆,¹⁸O) 3-hydroxy-3-phenylpropanoic acid, a putative intermediate in the biosynthesis of benzoic acid (BA) and salicylic acid (SA) from cinnamic acid, has been synthesized and administered to cucumber (Cucumis sativus L.) and Nicotiana attenuata (Torrey). Analysis of the products by gas chromatography-mass spectrometry revealed incorporation of labelling into BA and SA, but not into benzaldehyde. In a separate experiment, 3-hydroxy- 3-phenylpropanoic acid was found to be a metabolite of phenylalanine, itself the primary metabolic precursor of BA and SA. These data suggest that cinnamic acid chain shortening is probably achieved by β-oxidation, and that the proposed “non-oxidative” pathway of side-chain degradation does not function in the biosynthesis of BA and SA, in cucumber and N. attenuata. Received: 10 February 2000 / Accepted: 18 April 2000     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

Separation of bioactive biflavonoids from Rheedia gardneriana using chitosan modified with benzaldehyde

This paper shows the influence of benzenic groups on the chitosan surface for the separation of bioactive biflavonoids from Rheedia gardneriana leaves The yield of the biflavonoids using chitin modified with benzaldehyde (CH-Bz) as adsorbent in column chromatography was higher than that achieved with silica gel and chitosan. The presence of benzenic groups decreases the polarity of chitosan and consequently the interaction of hydrogen bonding between phenolic hydroxyl (OH) of biflavonoids and amine groups of the adsorbent. Therefore, the separation of these compounds appears to be the result of hydrophobicity and pi-pi interaction among electrons from the aromatic ring in sorbent and biflavonoid molecules.     

Environmental Contamination with Vancomycin-Resistant Enterococci from Hospital Sewage in Portugal

Vancomycin-resistant enterococci (VRE) were detected in samples of sewage obtained downstream of hospitals of the Porto area in Portugal, and in samples from the Douro Estuary. Clonal analysis, Tn1546 typing, and presence of putative virulence traits indicate the clinical origin of these isolates. This observation highlights the importance of hospital sewage in the VRE contamination of the environment.     

In vitro bi-enzymatic synthesis of benzaldehyde from phenylalanine: practical and mechanistic studies

The oxidation of phenylalanine to benzaldehyde was performed in vitro by a bi-enzymatic system d-amino acid oxidase-peroxidase. We describe attempts to optimise experimental conditions and we propose a probable mechanism for the transformation.     

Comparative tissue morphology in using prostheses from polypropylene and polytetrafluoretilen

To treat hernias, polymer net implants are widely used. We carried out a comparative study of the morphological picture of inflammation, cellular composition of tissues, and dynamics of scar formation in mice in using the implants Esfil and Uniflex under conditions of single and double introduction of cultivated fibroblasts and without fibroblasts in the area of disposition of the endoprostheses. A more expressed inflammatory reaction throughout the entire period of time was observed in implantation of the material Esfil. In using the material Uniflex, there was revealed a higher percent of fibroblasts than in using the Esfil prosthesis. Introduction of cultivated fibroblasts in using both materials modified the curve of dynamics of the inflammatory process by making it smoother. Use of the Uniflex endoprosthesis is preferable.     

Carotid artery mechanical properties and stresses quantified using in vivo data from normotensive and hypertensive humans

The goal of this study was to model the in vivo non-linear mechanical behavior of human common carotid arteries (CCAs) and then to compare wall stresses and associated contributions of micro-constituents in normotensive (NT) and treated hypertensive (HT) subjects. We used an established theoretical model of 3D arterial mechanics that assumes a hyperelastic, anisotropic, active–passive, and residually stressed wall. In vivo data were obtained non-invasively from CCAs in 16 NT (21–64 years old) and 25 treated HT (44–69 years old) subjects. The associated quasi-static boundary value problem was solved semi-analytically over a cardiac cycle while accounting for surrounding perivascular tissue. Best-fit values of model parameters, including those describing contributions by intramural elastin, fibrillar collagen, and vascular smooth muscle, were estimated by a non-linear least-squares method. The model (1) captured temporal changes in intraluminal pressure, (2) estimated wall stress fields that appeared to reflect the presence or absence of age and disease, and (3) suggested changes in mechanical characteristics of wall micro-constituents despite medical treatment of hypertension. For example, age was positively correlated with residual stresses and altered fibrillar collagen in NT subjects, which indirectly validated the modeling, and HT subjects had higher levels of stresses, increased smooth muscle tone, and a stiffer elastin-dominated matrix despite treatment. These results are consistent with prior reports on effects of age and hypertension, but provide increased insight into evolving contributions of cell and matrix mechanics to arterial behavior in vivo.     

Formation of mineral phosphate microtubes in the presence of halophilic cyanobacteriumMicrocoleus chthonoplastes

In a culture of filamentous cyanobacteriumMicrocoleus chthonoplastes under high phosphorus content and the presence of NaF, formation of aggregated microtubes of 0.8–1.0 m in diameter was obtained. Microtubes contain calcium and phosphorus. These filamentous structures do not represent fossilized bacteria.     

Contamination of exosome preparations,isolated from biological fluids

The aim of this study was to attract attention of researchers to the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 (JEOL, Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: blood plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and conditioned cell culture medium (MDCK, MDA-MB, and MCF-7 cells). All examined preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations were characterized by the presence of contaminating structures: low electron density particles without limiting membrane and therefore could not be attributed to exosomes (“non-vesicles”). Two main types of the “non-vesicles” were found in the exosome preparations: particles of 20–40 nm in size, representing 10–40% of all structures in the exosome preparations; and particles of 40–100 nm in size (identical to exosomes by size). Morphology of the “non-vesicles” corresponded to that of intermediate and low density lipoproteins (20–40 nm), and very low density lipoproteins (40–100 nm), which were identical to exosomes in their size. The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparations by electron microscopy and to take into consideration the presence of contaminating structures in the analysis of experimental data.     

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.     

Tissue stresses and strain in trabeculae of a canine proximal femur can be quantified from computer reconstructions

A quantitative assessment of bone tissue stresses and strains is essential for the understanding of failure mechanisms associated with osteoporosis, osteoarthritis, loosening of implants and cell-mediated adaptive bone-remodeling processes. According to Wolff's trajectorial hypothesis, the trabecular architecture is such that minimal tissue stresses are paired with minimal weight. This paradigm at least suggests that, normally, stresses and strains should be distributed rather evenly over the trabecular architecture. Although bone stresses at the apparent level were determined with finite element analysis (FEA), by assuming it to be continuous, there is no data available on trabecular tissue stresses or strains of bones in situ under physiological loading conditions. The objectives of this project were to supply reasonable estimates of these quantities for the canine femur, to compare trabecular-tissue to apparent stresses, and to test Wolff's hypothesis in a quantitative sense. For that purpose, the newly developed method of large-scale micro-FEA was applied in conjunction with micro-CT structural measurements. A three-dimensional high-resolution computer reconstruction of a proximal canine femur was made using a micro-CT scanner. This was converted to a large-scale FE-model with 7.6 million elements, adequately refined to represent individual trabeculae. Using a special-purpose FE-solver, analyses were conducted for three different orthogonal hip-joint loading cases, one of which represented the stance-phase of walking. By superimposing the results, the tissue stress and strain distributions could also be calculated for other force directions. Further analyses of results were concentrated on a trabecular volume of interest (VOI) located in the center of the head. For the stance phase of walking an average tissue principal strain in the VOI of 279 strain was found, with a standard deviation of 212 microstrain. The standard deviation depended not only on the hip-force magnitude, but also on its direction. In more than 95% of the tissue volume the principal stresses and strains were in a range from zero to three times the averages, for all hip-force directions. This indicates that no single load creates even stress or strain distributions in the trabecular architecture. Nevertheless, excessive values occurred at few locations only, and the maximum tissue stress was approximately half the value reported for the tissue fatigue strength. These results thus indicate that trabecular bone tissue has a safety factor of approximately two for hip-joint loads that occur during normal activities.     

Tissue stresses and strain in trabeculae of a canine proximal femur can be quantified from computer reconstructions

A quantitative assessment of bone tissue stresses and strains is essential for the understanding of failure mechanisms associated with osteoporosis, osteoarthritis, loosening of implants and cell- mediated adaptive bone-remodeling processes. According to Wolff's trajectorial hypothesis, the trabecular architecture is such that minimal tissue stresses are paired with minimal weight. This paradigm at least suggests that, normally, stresses and strains should be distributed rather evenly over the trabecular architecture. Although bone stresses at the apparent level were determined with finite element analysis (FEA), by assuming it to be continuous, there is no data available on trabecular tissue stresses or strains of bones in situ under physiological loading conditions. The objectives of this project were to supply reasonable estimates of these quantities for the canine femur, to compare trabecular-tissue to apparent stresses, and to test Wolff's hypothesis in a quantitative sense. For that purpose, the newly developed method of large-scale micro-FEA was applied in conjunction with micro-CT structural measurements. A three-dimensional high-resolution computer reconstruction of a proximal canine femur was made using a micro-CT scanner. This was converted to a large-scale FE-model with 7.6 million elements, adequately refined to represent individual trabeculae. Using a special-purpose FE-solver, analyses were conducted for three different orthogonal hip-joint loading cases, one of which represented the stance-phase of walking. By superimposing the results, the tissue stress and strain distributions could also be calculated for other force directions. Further analyses of results were concentrated on a trabecular volume of interest (VOI) located in the center of the head. For the stance phase of walking an average tissue principal strain in the VOI of 279 strain was found, with a standard deviation of 212 microstrain. The standard deviation depended not only on the hip-force magnitude, but also on its direction. In more than 95% of the tissue volume the principal stresses and strains were in a range from zero to three times the averages, for all hip-force directions. This indicates that no single load creates even stress or strain distributions in the trabecular architecture. Nevertheless, excessive values occurred at few locations only, and the maximum tissue stress was approximately half the value reported for the tissue fatigue strength. These results thus indicate that trabecular bone tissue has a safety factor of approximately two for hip-joint loads that occur during normal activities.