Purification of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and receptor binding

Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.     

Grubbing by wild boars (Sus scrofa L.) and its impact on hardwood forest soil carbon dioxide emissions in Switzerland

Interest in soil C storage and release has increased in recent years. In addition to factors such as climate/land-use change, vertebrate animals can have a considerable impact on soil CO₂ emissions. To date, most research has considered herbivores, while the impact of omnivorous animals has rarely been investigated. Our goal was to determine how European wild boars (Sus scrofa L.), large omnivores that consume soil-inhabiting animals and belowground plant parts by grubbing in the soil, affect soil C dynamics. We measured soil respiration (CO₂), temperature, and moisture on paired grubbed and non-grubbed plots in six hardwood forest stands for a 3-year period and sampled fine root and microbial biomass at the beginning and after 2 years of the study. We also measured the percentage of freshly disturbed forest soil within the larger surroundings of each stand and used this information together with hunting statistics and forest cover data to model the total amount of CO₂ released from Swiss forest soils due to grubbing during 1 year. Soil CO₂ emissions were significantly higher on grubbed compared to non-grubbed plots during the study. On average 23.1% more CO₂ was released from these plots, which we associated with potential alterations in CO₂ diffusion rates, incorporation of litter into the mineral soil and higher fine root/microbial biomass. Thus, wild boars considerably increased the small-scale heterogeneity of soil properties. Roughly 1% of Switzerland’s surface area is similar to our sites (boar density/forest cover). Given the range of forest soil disturbance of 27–54% at our sites, the geographic information system model predicted that boar grubbing would lead to the release of an additional 49,731.10–98,454.74 t CO₂ year⁻¹. These values are relatively small compared to total soil emissions estimated for Swiss hardwood forests and suggest that boars will have little effect on large-scale emissions unless their numbers increase and their range expands dramatically.     

Searching for monooxygenases and hydrolases in bacteria from an extreme environment

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.     

Exercise therapy and cognitive behavioural therapy to improve fatigue,daily activity performance and quality of life in Postpoliomyelitis Syndrome: the protocol of the FACTS-2-PPS trial

Background  

Postpoliomyelitis Syndrome (PPS) is a complex of late onset neuromuscular symptoms with new or increased muscle weakness and muscle fatigability as key symptoms. Main clinical complaints are severe fatigue, deterioration in functional abilities and health related quality of life. Rehabilitation management is the mainstay of treatment. Two different therapeutic interventions may be prescribed (1) exercise therapy or (2) cognitive behavioural therapy (CBT). However, the evidence on the effectiveness of both interventions is limited. The primary aim of the FACTS-2-PPS trial is to study the efficacy of exercise therapy and CBT for reducing fatigue and improving activities and quality of life in patients with PPS. Additionally, the working mechanisms, patients' and therapists' expectations of and experiences with both interventions and cost-effectiveness will be evaluated.     

An efficient system to generate monoclonal antibodies against membrane-associated proteins by immunisation with antigen-expressing mammalian cells

Background  

The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context.