Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

Subthalamic nucleus deep brain stimulation in elderly patients – analysis of outcome and complications

Background  

There is an ongoing discussion about age limits for deep brain stimulation (DBS). Current indications for DBS are tremor-dominant disorders, Parkinson's disease, and dystonia. Electrode implantation for DBS with analgesia and sedation makes surgery more comfortable, especially for elderly patients. However, the value of DBS in terms of benefit-risk ratio in this patient population is still uncertain.     

Pulmonary hypertension associated with sarcoidosis

Pulmonary involvement is common in sarcoidosis, an immune-mediated inflammatory disorder that is characterized by non-caseating granulomas in tissue. Sarcoid patients with advanced pulmonary disease, especially end-stage pulmonary fibrosis, risk developing pulmonary hypertension (World Health Organization group III pulmonary hypertension secondary to hypoxic lung disease). Increased levels of endothelin (ET)-1 in plasma and bronchoalveolar lavage of some sarcoid patients suggest that ET-1 may be driving pulmonary fibrosis and sarcoidosis-associated pulmonary hypertension. Although a relationship between raised levels of ET-1 and clinical phenotype is yet to be identified, early evidence from studies of ET-1 blockade with drugs such as bosentan is encouraging. Such therapy possibly could be combined with standard anti-inflammatory agents to improve outcome.     

Transposon mediated transgenesis in a marine invertebrate chordate: <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis.     

Variation in habitat choice and delayed reproduction: adaptive queuing strategies or individual quality differences?

In most species, some individuals delay reproduction or occupy inferior breeding positions. The queue hypothesis tries to explain both patterns by proposing that individuals strategically delay breeding (queue) to acquire better breeding or social positions. In 1995, Ens, Weissing, and Drent addressed evolutionarily stable queuing strategies in situations with habitat heterogeneity. However, their model did not consider the non-mutually exclusive individual quality hypothesis, which suggests that some individuals delay breeding or occupy inferior breeding positions because they are poor competitors. Here we extend their model with individual differences in competitive abilities, which are probably plentiful in nature. We show that including even the smallest competitive asymmetries will result in individuals using queuing strategies completely different from those in models that assume equal competitors. Subsequently, we investigate how well our models can explain settlement patterns in the wild, using a long-term study on oystercatchers. This long-lived shorebird exhibits strong variation in age of first reproduction and territory quality. We show that only models that include competitive asymmetries can explain why oystercatchers' settlement patterns depend on natal origin. We conclude that predictions from queuing models are very sensitive to assumptions about competitive asymmetries, while detecting such differences in the wild is often problematic.     

Nucleoside Map of the Human Central Nervous System

Nucleosides are neuromodulators that have a wide range of biological roles in the brain. In order to better understand the function of nucleosides in the human central nervous system (CNS), we constructed a nucleoside map showing the concentration of various nucleosides and their metabolites using post mortem samples from 61 human brain areas and 4 spinal cord areas. We evaluated in vivo tissue levels of four nucleosides (uridine, inosine, guanosine, and adenosine) and three of their metabolites (uracil, hypoxanthine, and xanthine). The concentrations of nucleosides were unevenly distributed across different brain regions, where the highest levels were found in the cerebral cortex and basal ganglia, whereas the lowest concentrations were located in the locus coeruleus, the zona incerta, the substantia nigra, and the inferior colliculus. The regional differences in nucleoside levels in the CNS may reflect the distinct physiological functions adopted by these compounds in different brain areas.     

Shoot organogenesis in elite clones of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

An efficient shoot organogenesis system has been developed from mature plants of selected elite clones of Eucalyptus tereticornis Sm. Cultures were established using nodal explants taken from freshly coppice shoots cultured on Murashige and Skoog medium containing 58 mM sucrose, 0.7% (w/v) agar (MS medium) and supplemented with 2.5 μM benzyladenine (BA) and 0.5 μM α-naphthaleneacetic acid (NAA). Shoot organogenesis was achieved from leaf segments taken from elongated microshoots on MS medium supplemented with 5.0 μM BA and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of cefotaxime to the medium promoted shoot differentiation, whereas carbenicillin and cephalexin inhibited shoot differentiation. Maximum shoot bud organogenesis (44.6%) occurred in explants cultured on MS medium supplemented with 5.0 μM BA, 1.0 μM 2,4-D and 500 mg/l cefotaxime. Leaf maturity influenced shoot regeneration, with maximum shoot organogeneisis (40.5%) occurring when the source of explants was the fifth leaf (14–16 days old) from the top of microshoot. Shoot organogenic potential also varied amongst the different clones of E. tereticornis. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses indicated clonal uniformity of the newly formed shoots/plants, and these were also found to be true-to-type.     

Conservation of the Native Orchids Through Seedling Culture and Reintroduction—A Singapore Experience

Singapore is located near the equator, off the southern tip of the Malay Peninsula. The whole country consists mostly of lowland. It has many interesting types of natural habitats such as primary rain forest, freshwater swamp forest, mangroves, secondary forests, shrub, grasslands, and urban parks and fields. The climate is equatorial with relatively uniform temperature and high humidity. Unfortunately, many of the natural habitats and the native orchids which thrive there have disappeared due to habitat destruction. Some 226 species of native orchids have been recorded in Singapore. However, of these 178 are considered to be extinct, and only five are common. The orchid conservation programme aims to monitor existing species, explore ways to conserve their germplasm, and increase their numbers in natural, semi-natural, and urban environments through ex-situ seedling culture and subsequent re-introduction into appropriate habitats, including roadside trees, parks and natural areas. In the first phase of the programme, we have successfully propagated and carried out experiments of re-introduction on five species of native orchids, namely, Grammatophyllum speciosum, Bulbophyllum vaginatum, Bulbophyllum membranaceum, Cymbidium finlaysonianum and Cymbidium bicolor. Survival percentages 8-yr after the reintroduction events ranged from 10 to 95 for G. speciosum, the target species of the earliest re-introduction experiments. Size of the seedlings at reintroduction, host trees, and relative humidity seemed to play significant roles in the success rate of the reintroductions.     

The zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>) impacts European bitterling (<Emphasis Type="Italic">Rhodeus amarus</Emphasis>) load in a host freshwater mussel (<Emphasis Type="Italic">Unio pictorum</Emphasis>)

The European bitterling, Rhodeus amarus, is a non-indigenous fish species in British fresh waters. It lays its eggs in unionid mussels which themselves are vulnerable to fouling by the non-indigenous zebra mussel, Dreissena polymorpha. Observations from an unmanipulated natural system showed that only 27% of zebra mussel-fouled Unio pictorum hosted bitterling, while 47% of unfouled U. pictorum hosted bitterling. We conducted a field experiment in the River Great Ouse catchment, Cambridgeshire, England in May–June 2007 and 2008 to quantify the impact of zebra mussels on bitterling load in host mussels. Zebra mussel-fouled unionids were significantly less likely to host bitterling than unfouled unionids. The number of unionids hosting bitterling did not differ significantly whether the zebra mussels fouling the unionid were alive or dead. Bitterling appeared to discriminate against zebra mussel-fouled unionids less as the 2007 breeding season advanced, potentially because preferred unfouled unionids had a higher bitterling load, and were therefore relatively lower quality hosts than at the start of the breeding season.