GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.     

Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein^?1 min^?1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP^?1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.     

Protein partition between the different phases comprising poly(ethylene glycol)-salt aqueous two-phase systems, hydrophobic interaction chromatography and precipitation: a generic description in terms of salting-out effects

The solution behaviour of selected proteins has been studied under conditions promoting precipitation, binding to mildly hydrophobic adsorbents or partition. Solvophobic theory may be used to describe these forms of protein partition. The tendency of a protein to partition therein is dependent upon surface properties of the protein solute mediated by the concentration and nature of added salts. As applied to partitioning in poly(ethylene glycol) (PEG)-salt systems this implies that linear (Brönsted) relationships apply only to proteins partitioned close to the critical point. At longer tie-line lengths protein partitioning is increasingly influenced by salting-out forces. This is confirmed by the observed behaviour of the proteins. The point at which this behaviour changes has been unambiguously defined enabling the direct comparison of phase transition of proteins during partition in all systems. The results obtained show that phase transition during adsorption and partition occur at similar concentrations of salt. This is less than that required to promote precipitation. It appears, from these limited studies, that top-phase preferring proteins are partitioned at salt concentrations above those required to cause adsorption. Proteins preferring the lower phase are partitioned at salt concentrations close to or below those required for adsorption. This raises questions regarding the solvated molecular form of the partitioned proteins and the definition of the partition coefficient.     

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase

Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml(-1), pH 7.8, temperature 37 degrees C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.9-0.99 mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and volumetric productivity was 3.6-4.6 micromol min(-1) ml(-1). In addition the product 6-APA could be directly crystallized from the top phase with a purity of 96%.     

Comparative morphology of spermatozoa and reproductive systems of zorapteran species from different world regions (Insecta,Zoraptera)

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Zorotypus huxleyi (Ecuador) and Zorotypus weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory glands. A long cuticular flagellum is present in Z. magnicaudelli and in the previously studied Zorotypus caudelli like in several other species, whereas it is absent in Z. weidneri, Z. huxleyi, Zorotypus hubbardi, Zorotypus impolitus and Zorotypus guineensis. Characteristic features of the very similar sperm are the presence of: a) two dense arches above the axoneme; b) a 9 + 9+2 axoneme with detached subtubules A and B of doublets 1 and 6; c) the axonemal end degenerating with enlarging accessory tubules; d) accessory tubules with 17 protofilaments; e) three accessory bodies beneath the axoneme; and f) two mitochondrial derivatives of equal shape. The first characteristic (a) is unknown outside of Zoraptera and possibly autapomorphic. The sperm structure differs distinctly in Z. impolitus and Z. hubbardi, which produce giant sperm and possess a huge spermatheca. The presence of the same sperm type in species either provided with a sclerotized coiled flagellum in males or lacking this structure indicates that a different organization of the genital apparatus does not necessarily affect the sperm structure. The flagellum and its pouch has probably evolved within Zoraptera, but it cannot be excluded that it is a groundplan feature and was reduced several times. The fossil evidence and our findings suggest that distinct modifications in the genital apparatus occurred before the fragmentation of the Gondwanan landmass in the middle Cretaceous.     

Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability

The biotech industry is, nowadays, facing unparalleled challenges due to the enhanced demand for biotechnology-based human therapeutic products, such as monoclonal antibodies (mAbs). This has led companies to improve substantially their upstream processes, with the yield of monoclonals increasing to titers never seen before. The downstream processes have, however, been overlooked, leading to a production bottleneck. Although chromatography remains the workhorse of most purification processes, several limitations, such as low capacity, scale-related packing problems, low chemical and proteolytic stability and resins' high cost, have arisen. Aqueous two-phase extraction (ATPE) has been successfully revisited as a valuable alternative for the capture of antibodies. One of the important remaining questions for this technology to be adopted by the biotech industries is, now, how it compares to the currently established platforms in terms of costs and environmental impact. In this report, the economical and environmental sustainability of the aqueous two-phase extraction process is evaluated and compared to the currently established protein A affinity chromatography. Accordingly, the ATPE process was shown to be considerably advantageous in terms of process economics, especially when processing high titer cell culture supernatants. This alternative process is able to purify continuously the same amount of mAbs reducing the annual operating costs from 14.4 to 8.5 million (US$/kg) when cell culture supernatants with mAb titers higher than 2.5 g/L are processed.     

Thermodynamic modeling of the partitioning of biomolecules in aqueous two-phase systems using a modified Flory–Huggins equation

A modified Flory–Huggins equation accounting for the solvation of polymer molecules by water molecules was used to model the phase behavior of aqueous two-phase systems (ATPS) formed by poly(ethylene glycol) (PEG) and dextran. The parameters of the equation were obtained by fitting experimental equilibrium data either accounting for or disregarding dextran polidispersity. The modified equation was subsequently applied to calculate partition coefficients of biomolecules in these systems. It was found that accounting for polidispersity did not affect significantly the calculated phase equilibrium, but increased the agreement of calculated partition coefficients with experimental data. Further improvement was obtained by using a size dependent interaction parameter for dextran pseudo-components.     

Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.     

Hydrolysis of triglyceride by the whole cell of Pseudomonas putida 3SK in two-phase batch and continuous reactors systems

Batch and continuous hydrolysis of olive oil in an organic-aqueous two-phase system using the live whole cell of Pseudomonas putida 3SK as a source of a lipase is investigated. The strain was not only fully viable and grown well, but also produced extracellular lipase simultaneously. The degree of hydrolysis, depending on olive oil concentration in the solvents, was maximal at 13.5% (w/v) and decreased with the increase of the substrate concentration. At the optimal condition, a degree of hydrolysis higher than 95% was achieved with 24 h at 30 degrees C when the reaction was carried out in a two-phase batch stirred reactor. For long-term operation a continuous stirred reactor was designed. When the reaction was carried out in a continuous stirred reactor, the degree was hydrolysis reached 86% at a dilution rate of 0.2 h(-1). Satisfactory performance of a two-phase bioreactor was obtained in a long-term continous operation, which lasted for at least 30 days by feeding organic solvent containing olive oil and aqueous media separately. (c) 1994 John Wiley & Sons, Inc.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

Aggregation and denaturation of antibodies: a capillary electrophoresis, dynamic light scattering, and aqueous two-phase partitioning study

Protein denaturation and aggregation are well-known problems in the pharmaceutical industry. As the protein aggregates, it loses its biological activity and creates problems in its administration to patients. In this paper, we explore the use of aqueous two-phase systems, capillary zone electrophoresis, and dynamic light scattering for the monitoring of protein denaturation and aggregation. Our studies focus on human IgG and HSA. Capillary zone electrophoresis was used to monitor changes in the charge to size ratio of the proteins upon denaturation and dynamic light scattering was used to detect the presence of any aggregates and to monitor the size of the proteins. The information obtained from aqueous two-phase partitioning is similar to that obtained from capillary zone electrophoresis. The simplicity of aqueous two-phase system and its low cost (compared to the other analytical techniques) suggest that it can be routinely used for the quality control of some pharmaceutical preparations.     

Effects of three benzimidazoles on growth, general morphology and ultrastructure of Tritrichomonas foetus

Tritrichomonas foetus is a venereal pathogen of cattle, which causes infertility, early embryonic death or abortion. In order to evaluate the potential trichomonicidal activity of benzimidazoles, the effects of thiabendazole, mebendazole and albendazole were analyzed on the multiplication, general morphology and ultrastructure of T. foetus. It was found that mebendazole presented the highest IC(50%) (2.3 microM), when compared with albendazole (IC(50%)=9.4 microM) and thiabendazole (IC(50%)=142.6 microM), and that such effects were irreversible. Concerning microscopic analysis, thiabendazole- and mebendazole-treated cells presented increased volume, internalization of the flagella, disruption or multiplication of the nucleus, multiple organelles and cytoplasmic vacuolization. Albendazole-treated cells exhibited slight alterations, because the parasite became slightly rounded, its flagella were not internalized but the cytoplasm was vacuolated. Mebendazole was indeed highly effective as an in vitro trichomonicidal agent, and this might open up new possibilities for the use of mebendazole in the therapy of bovine trichomoniasis.     

Extraction of β-galactosidase fused protein a in aqueous two-phase systems

A method for the purification of proteins hybridized with β-galactosidase and produced in Escherichia coli is suggested. The method is based on the dominating properties of the β-galactosidase part of the molecule that are utilized for extraction in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system. The purification of the hybrid protein Staphylococcal protein A-Escherichia coli-β-galactosidase (SpA-βgal) produced in Escherichia coli is described. The partitioning of the cell debris and SpA-βgal depended on the distance to the critical point, i.e., the length of the tie line. A poly(ethylene glycol) top phase and an interface free from cell debris were obtained for a composition close to the binodial with a relatively short tie line. At this composition no Spa-βgal partitioned to the interface. When the length of the tie line was increased, more of the SpA-βgal was caught by the interface. The partitioning of SpA-βgal to the top phase was also affected by the salts present during the extraction. The utilization of SpA-βgal for affinity extraction has been investigated. Experiments with SpA-βgal and fluorescence-labeled human IgG(hIgG-F) in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system showed that the complex SpA-βgal-hlgG-F was partitioned to the interface, probably as a precipitate.     

On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins

The behavior of a series of pure proteins partitioned in aqueous two-phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid-liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two-phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. (c) 1994 John Wiley & Sons, Inc.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Affinity gel electrophoresis as a predictive technique in the fractionation of transgenic sheep milk proteins by affinity aqueous two-phase partitioning

Affinity electrophoresis in the presence of various triazine dyes of sheep milk proteins, including transgenically-introduced human ₁-antitrypsin, has been evaluated as a predictive technique for possible large scale affinity-driven aqueous two-phase purifications. The success of the approach suggested it has potential as a general method for the rapid screening of ligands using only g amounts of sample, that could be applied to many complex mixtures before embarking on more costly and time-consuming two-phase partitioning experiments.     

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues

BacKGROUND AND AIMS: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. METHODS: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. KEY RESULTS: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. CONCLUSIONS: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.     

Fractionation in two-phase systems of red cells during rat development: Changes in pyruvate kinase and bisphosphoglycerate mutase activities in relation to red cell switching

Summary An inverse relationship between 2,3-bisphosphoglycerate levels and the ratio calculated from pyruvate kinase and bisphosphoglycerate mutase activities has been observed in red populations of rats during animal development. Counter-current distribution in aqueous two-phase systems of these cells populations shows a displacement of distribution profiles towards the high-numbered cavities of the rotor as animal ages. Heterogeneity of cells after distribution is only observed during the switching process from fetal to adult red cells taking place along the postnatal stage of development. Values for the pyruvate kinase/bisphosphoglycerate mutase ratio in these fractions suggest the separation of fetal (liver) from adult (bone marrow) red cells.     

Antimicrobial activity of AOT-isooctane reverse micelle as a bioseparation and biocatalysis tool

Abstract

The antimicrobial activity of different reverse micelles on microorganisms is been compared using the disc diffusion method. The bis (2-ethylhexyl) sodium sulfosuccinate (AOT) reverse micelle showed a more significant inhibitory effect than do other reverse. micelles, and it had an antimicrobial activity against a broad range of microorganisms. Results from an antimicrobial activity test of isooctane and a forward extraction containing soybean protein suggest that the surfactant was chiefly responsible for inhibiting microbes in AOT/isooctane reverse micelle, while isooctane hardly inhibited the microbial growth. The properties of S. aureus, cultured in the TSB with AOT reverse micellar solution, were identified by the SEM and SDS-PAGE fingerprinting of cell-wall proteins. It is concluded that the cell-wall of the S. aureus decreased in the TSB with AOT reverse micellar solution, and some cell protein subunits of the S. aureus did not occurr, especially between 14.4 and 42.7 kDa, while one new protein subunit at near 97.4 kDa occurred