The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila

Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity.     

Molecular anatomy of a trafficking organelle

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.     

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.     

Observing local and global properties of metabolic pathways: 'load points' and 'choke points' in the metabolic networks

MOTIVATION: The local and global aspects of metabolic network analyses allow us to identify enzymes or reactions that are crucial for the survival of the organism(s), therefore directing us towards the discovery of potential drug targets. RESULTS: We demonstrate a new method ('load points') to rank the enzymes/metabolites in the metabolic network and propose a model to determine and rank the biochemical lethality in metabolic networks (enzymes/metabolites) through 'choke points'. Based on an extended form of the graph theory model of metabolic networks, metabolite structural information was used to calculate the k-shortest paths between metabolites (the presence of more than one competing path between substrate and product). On the basis of these paths and connectivity information, load points were calculated and used to empirically rank the importance of metabolites/enzymes in the metabolic network. The load point analysis emphasizes the role that the biochemical structure of a metabolite, rather than its connectivity (hubs), plays in the conversion pathway. In order to identify potential drug targets (based on the biochemical lethality of metabolic networks), the concept of choke points and load points was used to find enzymes (edges) which uniquely consume or produce a particular metabolite (nodes). A non-pathogenic bacterial strain Bacillus subtilis 168 (lactic acid producing bacteria) and a related pathogenic bacterial strain Bacillus anthracis Sterne (avirulent but toxigenic strain, producing the toxin Anthrax) were selected as model organisms. The choke point strategy was implemented on the pathogen bacterial network of B.anthracis Sterne. Potential drug targets are proposed based on the analysis of the top 10 choke points in the bacterial network. A comparative study between the reported top 10 bacterial choke points and the human metabolic network was performed. Further biological inferences were made on results obtained by performing a homology search against the human genome. AVAILABILITY: The load and choke point modules are introduced in the Pathway Hunter Tool (PHT), the basic version of which is available on http://www.pht.uni-koeln.de.     

Renal function of gene-targeted mice lacking both SGK1 and SGK3

Serum- and glucocorticoid-inducible kinase (SGK) 1 and SGK3 share the ability to upregulate several ion channels, including the epithelial Na(+) channel. Whereas SGK1 is under genomic control of mineralocorticoids and glucocorticoids, SGK3 is constitutively expressed. The SKG1-knockout (sgk1(-/-)) mouse is seemingly normal when it is fed a standard diet, but its ability to retain NaCl is impaired when it is fed a salt-deficient diet. In the SGK3-knockout (sgk3(-/-)) mouse fed standard and salt-deficient diets, hair growth is strikingly delayed but NaCl excretion is normal. Thus the possibility was considered that SGK1 and SGK3 could mutually replace each other, thus preventing severe NaCl loss in sgk1(-/-) and sgk3(-/-) mice. We crossed SGK1- and SGK3-knockout mice and compared renal electrolyte excretion of the double mutants (sgk1(-/-)/sgk3(-/-)) with that of their wild-type littermates (sgk1(+/+)/sgk3(+/+)). Similar to sgk3(-/-) mice, the sgk1(-/-)/sgk3(-/-) mice display delayed hair growth. Blood pressure was slightly, but significantly (P < 0.03), lower in sgk1(-/-)/sgk3(-/-) (102 +/- 4 mmHg) than in sgk1(+/+)/sgk3(+/+) (114 +/- 3 mmHg) mice, a difference that was maintained in mice fed low- and high-salt diets. Plasma aldosterone concentrations were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) than in sgk1(+/+)sgk3(+/+) mice fed control (511 +/- 143 vs. 143 +/- 32 pg/ml) and low-salt (1,325 +/- 199 vs. 362 +/- 145 pg/ml) diets. During salt depletion, absolute and fractional excretions of Na(+) were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) (1.2 +/- 0.2 micromol/24 h g body wt, 0.12 +/- 0.03%) than in sgk1(+/+)/sgk3(+/+) (0.4 +/- 0.1 micromol/24 h g body wt, 0.04 +/- 0.01%) mice. The sgk1(-/-)/sgk3(-/-) mice share the delayed hair growth with sgk3(-/-) mice and the modestly impaired renal salt retention with sgk1(-/-) mice. Additional lack of the isoform kinase does not substantially compound the phenotype for either property.     

Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminichthys (Teleostei: Cyprinidae)

The Dalmatian cyprinid genus Phoxinellus is characterized by reductive characters most likely associated with the environmental conditions of small karstic streams, where all species of this genus occur. Based on 33 morphological traits, nuclear and mtDNA sequences Phoxinellus was found to be paraphyletic and included three not closely related monophyletic units. The scientific name Phoxinellus should therefore be restricted to species having plain coloration, small or absent postcleithrum, no genital papilla and an almost entirely naked body such as P. alepidotus, P. dalmaticus, and P. pseudalepidotus. Species that also have a small or absent postcleithrum and no genital papilla but display a dark stripe from the head to the caudal peduncle, and are entirely covered by distinct, not overlapping scales should be positioned closely to Telestes. Thus, we suggest inclusion of Phoxinellus croaticus, P. fontinalis and Paraphoxinus metohiensis in the genus Telestes. The Phoxinellus species that have a irregularly spotted color pattern, a large postcleithrum, an increased number of precaudal anal-fin pterygiophores, and a large genital papilla in females represent its own evolutionary line closely related to the Balkan species of Pseudophoxinus. For this monophyletic group, we propose to introduce a new genus: Delminichthys. This genus includes the species D. adspersus, D. ghetaldii, D. krbavensis and D. jadovensis.     

Phylogenetic relationships in the ‘<Emphasis Type="Italic">Pinnatella</Emphasis>’ clade of the moss family Neckeraceae (Bryophyta)

The family Neckeraceae is composed of three distinct clades, of which two, i.e. the Neckera and Thamnobryum clades, are well defined. The third clade, consisting of species belonging to Caduciella, Curvicladium, Handeliobryum, Himantocladium, Homaliodendron, Hydrocryphaea, Neckera, Neckeropsis, Pinnatella, Shevockia and Taiwanobryum, is the focus of this study. Based on sequence data from the trnS-rps4-trnT-trnL-trnF plastid cluster and the rpl16 intron as well as from nuclear ITS1&2, the phylogenetic relationships of these genera are reconstructed. The nearest relatives of this clade are resolved shedding more light on the evolution of the family. The generic composition of the clade and its individual genera are discussed; polyphyly requires redefinition of Pinnatella, Neckeropsis and Homaliodendron. The positions of Touwia and Homalia within the family are addressed in an additional analysis based on more extensive sequence data, and the corresponding new combinations are made. Several further taxonomic changes are proposed, including Circulifolium gen. nov., comprising the former Homaliodendron exiguum and H. microdendron.     

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from <Emphasis Type="Italic">Trametes multicolor</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d⁺, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d⁺ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d⁺ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d⁺ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h^-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.     

Energy dependence of radiochromic dosimetry films for use in radiotherapy verification

Aim

The purpose of the study was to examine the energy dependence of Gafchromic EBT radiochromic dosimetry films, in order to assess their potential use in intensity-modulated radiotherapy (IMRT) verifications.

Materials and methods

The film samples were irradiated with doses from 0.1 to 12 Gy using photon beams from the energy range 1.25 MeV to 25 MV and the film response was measured using a flat-bed scanner. The samples were scanned and the film responses for different beam energies were compared.

Results

A high uncertainty in readout of the film response was observed for samples irradiated with doses lower than 1 Gy. The relative difference exceeds 20% for doses lower than 1 Gy while for doses over 1 Gy the measured film response differs by less than 5% for the whole examined energy range. The achieved uncertainty of the experimental procedure does not reveal any energy dependence of Gafchromic EBT film response in the investigated energy range.

Conclusions

Gafchromic EBT film does not show any energy dependence in the conditions typical for IMRT but the doses measured for pre-treatment plan verifications should exceed 1 Gy.