Curation of the Plasmodium falciparum genome

The malaria genome has proved invaluable to researchers worldwide in the continuing fight against malaria by stimulating and underpinning molecular approaches in gene expression studies, vaccine and drug discovery research, and by providing data to facilitate hypothesis-driven research. The combination of in silico and experimental investigations has already yielded dividends by strengthening our understanding of the many facets of the malaria parasite Plasmodium falciparum. The recently initiated curation of the genome resource is a vital investment for maintaining and enhancing the use of this genomic information in the post-genomic era.     

Stemwood volume increment changes in European forests due to climate change—a simulation study with the EFISCEN model

This paper presents the results of a modelling study of future net annual increment changes in stemwood of European forests owing to climate change. Seven process‐based growth models were applied to 14 representative forest sites across Europe under one climate change scenario. The chosen scenario was the HadCM2 run, based on emission scenario IS92a, and resulted in an increase in mean temperature of 2.5 °C between 1990 and 2050, and an increase in annual precipitation of 5–15%. The information from those runs was incorporated in a transient way in a large‐scale forest resource scenario model, EFISCEN (European forest information scenario). European scale forest resource projections were made for 28 countries covering 131.7 million ha of forest under two management scenarios for the period until 2050. The results showed that net annual increments in stemwood of European forests under climate change will further increase with an additional 0.9 m³ ha^?1 y^?1 in 2030 compared to the ongoing increase under a current climate scenario, i.e. an extra 18% increase. After 2030 the extra increment increase is reduced to 0.79 m³ ha^?1 y^?1 in 2050. Under climate change, absolute net annual increments will increase from the present 4.95, on average for Europe, to 5.93 m³ ha^?1 y^?1 in 2025. After 2025, increments in all scenarios start to decline owing to ageing of the forest and the high growing stocks being reached. The results of the present study are surrounded by large uncertainties. These uncertainties are caused by unknown emissions in the future, unknown extent of climate change, uncertainty in process‐based models, uncertainty in inventory data, and uncertainty in inventory projection. Although the results are thus not conclusive, climate change may lead to extra felling opportunities in European forests of 87 million m³y^?1. Because Europe's forests are intensively managed already, management may adapt to climate change relatively easily. However, this study also indicates that climate change may lead to a faster build‐up of growing stocks. That may create a less stable forest resource in terms of risks to storm damage.     

A sublocus of the multicopy microsatellite marker CMS1 maps proximal to spinal muscular atrophy (SMA) as shown by recombinant analysis

The critical region containing the spinal muscular atrophy (SMA) gene is flanked by the 5q11–q13 markers, D5S435 and D5S557, as determined by linkage analysis. Here we present the results of an analysis of a Dutch SMA family with the multicopy microsatellite marker CMS1. A crossover is revealed in the critical SMA region. We conclude that at least one of the CMS1 subloci maps proximal to the SMA gene. This reduces the minimal SMA region from approximately 1.4 Mb to 600–700 kb.     

The expanding small heat-shock protein family,and structure predictions of the conserved “α-crystallin domain”

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and -crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous -crystallin domain, which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic -sheet-rich motifs, connected by a hydrophilic -helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.Correspondence to: W.W. de Jong     

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.     

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

Background  

Methodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.     

Novel Protein-Protein Interactions Inferred from Literature Context

We have developed a method that predicts Protein-Protein Interactions (PPIs) based on the similarity of the context in which proteins appear in literature. This method outperforms previously developed PPI prediction algorithms that rely on the conjunction of two protein names in MEDLINE abstracts. We show significant increases in coverage (76% versus 32%) and sensitivity (66% versus 41% at a specificity of 95%) for the prediction of PPIs currently archived in 6 PPI databases. A retrospective analysis shows that PPIs can efficiently be predicted before they enter PPI databases and before their interaction is explicitly described in the literature. The practical value of the method for discovery of novel PPIs is illustrated by the experimental confirmation of the inferred physical interaction between CAPN3 and PARVB, which was based on frequent co-occurrence of both proteins with concepts like Z-disc, dysferlin, and alpha-actinin. The relationships between proteins predicted by our method are broader than PPIs, and include proteins in the same complex or pathway. Dependent on the type of relationships deemed useful, the precision of our method can be as high as 90%. The full set of predicted interactions is available in a downloadable matrix and through the webtool Nermal, which lists the most likely interaction partners for a given protein. Our framework can be used for prioritizing potential interaction partners, hitherto undiscovered, for follow-up studies and to aid the generation of accurate protein interaction maps.     

Tripolyphosphatase from Methanobacterium thermoautotrophicum (strain ΔH)

Abstract A low-melecular-mass polyphosphatase (tripolyphosphatase, PPPi) from the archaeon Methanobacterium thermoautotrophicum (strain ΔH) was purified 340-fold and characterized. The tripolyphosphatase showed an optimal activity at pH 9.7 (at 60°C). Though the highest activities were measured with tripolyphosphate, tetrapolyphosphate (57%), phosphate glass type 5 (41%) and phosphate glass type 15 (20%) could also be used as substrates. However, tripolyphosphatase was unable to use pyrophosphate. The enzyme was dependent on the presence of Mg²⁺. In the presence of 2 mM PPPi, an optimal activity was found at 6 mM Mg²⁺. The K _m for PPPi was estimated at 0.37 mM. In addition, the enzyme was inhibited by KF (50% at 6 mM) and appeared to be very heat stable: after an incubation of 2 h at 85°C about 85% of the activity was still present.