The ABC's (and XYZ's) of peptide sequencing

Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens. The key - but little understood - technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.     

The Arabidopsis GNOM ARF-GEF mediates endosomal recycling,auxin transport,and auxin-dependent plant growth

Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.     

Proteomic analysis of a rat pancreatic stellate cell line using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Pancreatic stellate cells (PaSC) are emerging as key mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of quiescent to activated PaSC may have a significant influence on the development of chronic pancreatitis. We aim to compare differentially expressed proteins in activated and serum-starved non-proliferating PaSC using a mass spectrometry-based proteomics strategy. We cultured an immortalized rat PaSC cell line in media supplemented with 10% fetal bovine serum and in serum-free media. Using gel-based mass spectrometry (GeLC-MS/MS), we identified nearly 1500 proteins. Qualitative and quantitative proteomic analysis revealed several hundred proteins as differentially abundant between the two cell states. Proteins of greater abundance in activated PaSC included isoforms of actin (e.g., smooth muscle actin) and ribosomal proteins. Conversely, proteins more abundant in non-proliferating PaSC than in activated PaSC included signaling proteins MAP kinase 3 and Ras-related proteins. In addition, we have determined the molecular functions and biological pathways for these proteins. We are confident that the application of mass spectrometry-based strategies, such as that described herein, to investigate specific proteins in PaSC may lead to a better understanding of the molecular mechanisms involved in pancreatic diseases, such as chronic pancreatitis.     

A multi-locus chloroplast phylogeny for the Cucurbitaceae and its implications for character evolution and classification

Cucurbitaceae contain c. 800 species in 130 genera and are among the economically most important families of plants. We inferred their phylogeny based on chloroplast DNA sequences from two genes, one intron, and two spacers (rbcL, matK, trnL, trnL-trnF, rpl20-rps12) obtained for 171 species in 123 genera. Molecular data weakly support the traditional subfamilies Cucurbitoideae (111 genera) and Nhandiroboideae (19 genera, 60 species), and recover most of the eleven tribes, but almost none of the subtribes. Indofevillea khasiana is sister to all other Cucurbitoideae, and the genera of Joliffieae plus a few Trichosantheae form a grade near the base of Cucurbitoideae. A newly discovered large clade consists of the ancestrally Asian genera Nothoalsomitra, Luffa, Gymnopetalum, Hodgsonia, Trichosanthes, and the New World tribe Sicyeae. Genera that are poly- or paraphyletic include Ampelosicyos, Cucumis, Ibervillea, Neoachmandra, Psiguria, Trichosanthes, and Xerosicyos. Flower characters, especially number of free styles, fusion of filaments and/or anthers, tendril type, and pollen size, exine, and aperture number correlate well with the chloroplast phylogeny, while petal and fruit characters as well as karyotype exhibit much evolutionary flexibility.     

Computer-assisted evaluation of kinematics of the two bundles of the anterior cruciate ligament.

The aim of this cadaveric study was to describe the kinematics of the anterior cruciate ligament (ACL)-intact, posterolateral (PL) bundle-deficient and ACL-deficient knee by applying a protocol for computer-assisted evaluation of knee kinematics. The hypothesis that the PL bundle functions mainly at low knee flexion angles was tested. An optical tracking system was used to acquire knee joint motion on 10 knees during clinical evaluations by tracking markers rigidly attached to the bones. The protocol included acquisition of anterior-posterior (AP) translations and internal-external (IE) rotations, and evaluation of three clinical knee laxity tests (anterior drawer, manual and instrumented Lachman). The data demonstrated no significant contribution to AP translation and IE laxity from the PL bundle over the entire range of motion. The clinical knee laxity tests showed no significant differences between the ACL-intact and PL bundle-deficient states. The hypothesis could not be proven. Current clinical knee laxity measurements may not be suited for detecting subtle changes such as PL bundle deficiency in the ACL anatomy. The computation of knee laxity might be a step towards a more precise kinematic test of knee stability not only in the native and torn ACL state of the knee but also in the reconstructed knee.     

Analysing ecological dynamics with relational event models: The case of biological invasions

Aims

Spatio-temporal processes play a key role in ecology, from genes to large-scale macroecological and biogeographical processes. Existing methods studying such spatio-temporally structured data either simplify the dynamic structure or the complex interactions of ecological drivers. The aim of this paper is to present a generic method for ecological research that allows analysing spatio-temporal patterns of biological processes at large spatial scales by including the time-varying variables that drive these dynamics.

Location

Global analysis at the level of 272 regions.

Methods

We introduce a method called relational event modelling (REM). REM relies on temporal interaction dynamics that encode sequences of relational events connecting a sender node to a recipient node at a specific point in time. We apply REM to the spread of alien species around the globe between 1880 and 2005, following accidental or deliberate introductions into geographical regions outside of their native range. In this context, a relational event represents the new occurrence of an alien species given its former distribution.

Results

The application of relational event models to the first reported invasions of 4835 established alien species outside of their native ranges from four major taxonomic groups enables us to unravel the main drivers of the dynamics of the spread of invasive alien species. Combining the alien species first records data with other spatio-temporal information enables us to discover which factors have been responsible for the spread of species across the globe. Besides the usual drivers of species invasions, such as trade, land use and climatic conditions, we also find evidence for species-interconnectedness in alien species spread.

Conclusions

Relational event models offer the capacity to account for the temporal sequences of ecological events such as biological invasions and to investigate how relationships between these events and potential drivers change over time.     

Molecular cytogenetic characterisation of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale)

The terminal heterochromatic segments of the long arms of 20 rye B-chromosomes were isolated by means of laser microdissection technology. Also the remaining portions of the long arms, along with the short arms of the same chromosomes were isolated. Each sample was used for degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplification reactions. The resulting products were used as probes for chromosome in situ hybridisation experiments, and in Southern hybridisation to digests of 0B and +B DNA. Competition hybridisation of these probes with 0B DNA allowed the detection of B-specific sequences. The terminal heterochromatin of the rye B-chromosome contains both B-specific sequences and sequences also present on the A-chromosomes of rye. The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. Primers designed to the cloned sequence (E1100) were used to search for related low copy sequences in 0B DNA. The sequences of the PCR products revealed no similarities to that of the clone E1100 except for the primer sequences. The possible origin of this sequence is discussed in the context of models for the evolution of the rye B-chromosome. EMBL and Genbank accession numbers: Z54196 (E1199): Z54278 (B1) Edited by: R. Appels