Sonic hedgehog is required for vascular outgrowth in the hindbrain choroid plexus

Critical to the exchange and metabolic functions served by tissues like brain choroid plexi and lung is the coherent development of an epithelial sheet of large surface area in tight apposition to an extensive vascular bed. Here, we present functional experiments in the mouse demonstrating that Sonic hedgehog (Shh) produced by hindbrain choroid plexus epithelium induces the extensive vascular outgrowths and vascular surface area fundamental to choroid plexus functions, but does not induce the more specialized endothelial cell features of fenestrations and bore size. Our findings indicate that these Shh-dependent vascular elaborations occur even in the presence of Vegf and other established angiogenic factors, suggesting either that the levels of these factors are inadequate in the absence of Shh or that a different set of factors may be more essential to choroid plexus outgrowth. Transducing the Shh signal is a perivascular cell—the pericyte—rather than the more integral vascular endothelial cell itself. Moreover, our findings suggest that hindbrain choroid plexus endothelial cells, as compared to other vascular endothelial cells, are more dependent upon pericytes for instruction. Thus, in addition to Shh acting on the progenitor pool for choroid plexus epithelial cells, as previously shown, it also acts on choroid plexus pericytes, and together serves the important role of coordinating the development of two disparate yet functionally dependent structures—the choroid plexus vasculature and its ensheathing epithelium.     

Real time monitoring of acylations during solid phase peptide synthesis: a method based on electrochemical detection

Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent.     

Sensory and pulmonary irritation of inhaled n-butylamine in CF-1 and NMRI mice

Sensory and pulmonary irritation of butylamine was investigated in CF-1 and NMRI mice according to the American standard test method (ASTM E981-84). The method is based on the reflexively induced reduction of the respiratory rate of mice, when exposed to chemical irritants. Sensory irritation was investigated in normal mice, yielding RD50 values (concentration which reduces the respiratory rate by 50%) of 121 and 246 ppm for CF-1 and NMRI mice, respectively. The concentration-effect curves were parallel, but had significantly different elevations, indicating a lower sensitivity of NMRI mice. Pulmonary irritation was investigated in mice, inhaling through a tracheal cannula, yielding RD50 values of 300 and 362 ppm for CF-1 and NMRI mice, respectively. No statistically significant difference between either the slopes or the elevations of the concentration-effect curves was found, indicating the same level of sensitivity of CF-1 and NMRI mice regarding pulmonary irritation. It can be concluded that the 2 mice stocks gave qualitatively comparable responses, but regarding sensory irritation they responded differently quantitatively. Thus for sensory irritation investigations the RD50 values obtained with NMRI mice should be multiplied by 0.49 to obtain comparable values to those, expected in the recommended stock given by E981-84.     

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.     

Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase

Transfer of potato tubers to low temperature leads after 2–4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70–80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m^–3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70–80% decrease of SPS expression resulted in a reproducible but non-proportional (10–40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo . Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.     

Comparing different methods for trapping mated queens of weaver ants (Oecophylla longinoda; Hymenoptera: Formicidae)

The predatory efficiency of African weaver ants Oecophylla longinoda and their utilisation in protein production is a function of ant abundance. Reliable control of insect pests in tropical crops is achieved when ant populations are constantly high. Transplanted populations of weaver ant colonies containing egg-laying queens are more stable than those without. Achieving such stability through collection of colonies established in the wild is usually difficult because of uncertainty in locating the nest containing the egg-laying queen. In this study, we investigated four methods that may be used to collect mated queens that subsequently can be used to stock ant nurseries. The catch efficiencies of (1) leaf traps, (2) paper traps (both types providing a refuge for founding queens), (3) random search for queens and (4) light trapping were compared. Light trapping was the most efficient way to collect queens followed by leaf traps, random search and, last, paper traps. Light trapping and random search, though, required the presence of a person throughout the ant's mating season (several months), whereas this was not required when using leaf and paper traps.     

A New Synthetic Approach Towards α- and β-LNA (Locked Nucleic Acids)

Abstract

A bicyclo[2.2.1] phenyl thioglycoside was efficiently synthesised and introduced as the key synthon in a novel method for convergent synthesis of β-LNA-nucleosides as well as their α-configurated isomers. An acid-induced ring-opening reaction on the corresponding bicyclo[2.2.1] methyl furanoside is also described.     

Identifying QTL and genetic correlations between fur quality traits in mink (Neovison vison)

Mapping of QTL affecting fur quality traits (guard hair length, guard hair thickness, density of wool, surface of the fur and quality) and skin length was performed in a three‐generation mink population (F₂ design). In the parental generation, Nordic Brown mink were crossed reciprocally with American Black short nap mink. In all, 1082 mink encompassing three generations were used for the analyses. The mink were genotyped for 104 microsatellites covering all 14 autosomes. The QTL analyses were performed by least‐square regression implemented in gridqtl software. Genetic and phenotypic correlations and heritabilities were estimated using the average information‐restricted maximum‐likelihood method. Evidence was found for QTL affecting fur quality traits on nine autosomes. QTL were detected for guard hair thickness on chromosomes 1, 2, 3, 6 and 13; for guard hair length on chromosomes 2, 3 and 6; for wool density on chromosomes 6 and 13; for surface on chromosomes 7, 12 and 13; for quality on chromosomes 6, 7, 11 and 13; and for skin length on chromosomes 7 and 9. Proximity of locations of QTL for guard hair length, guard hair thickness and for wool density and quality suggests that some of the traits are in part under the influence of the same genes. Traits under the influence of QTL at close or identical positions also were traits that were strongly genotypically correlated. Based on the results of correlation analyses, the most important single traits influencing the quality were found to be density of wool, guard hair thickness and appearance of the surface.