parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.     

Genomics of steroid hormones: in silico analysis of nucleotide sequence variants (polymorphisms) of the enzymes involved in the biosynthesis and metabolism of steroid hormones

Alterations of steroid hormone biosynthesis and metabolism are suspected to be involved in the pathogenesis of several diseases. Several polymorphisms of the enzymes involved in these processes have already been described and some could be associated with certain diseases. We attempted to examine the sequence variants of these genes in order to find novel variants by an in silico analysis. We analyzed the known human nucleotide sequences of the enzymes p450 side-chain cleavage enzyme, steroid 17-alpha-hydroxylase/17,20-lyase, 3-beta-hydroxysteroid dehydrogenase types 1 and 2, 21-hydroxylase, 11-beta-hydroxylase, aldosterone synthase, aromatase, 11-beta-hydroxysteroid dehydrogenase types 1 and 2, steroid 5-alpha-reductase types 1 and 2, steroid 5-beta-reductase, dehydroepiandrosterone sulfotransferase, 17-beta-hydroxysteroid dehydrogenase types 1–3. The analysis was performed using the National Center for Biotechnology Information Database by the search tool blastn. We found numerous sequence variants in both coding and non-coding sequences. The majority of these sequence variants have already been described, nevertheless, some appear as novel variants. Some of these may also have functional significance. We hypothesize over the possible significance of these findings and briefly review the available literature.     

Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer

BACKGROUND: Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1-10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method. METHODS: Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.ResultsTo increase the accuracy of FCET measurements, we applied a long wavelength donor-acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency. CONCLUSIONS: We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur.     

Supramolecular exciton chirality of carotenoid aggregates

The conventional organic chemistry concept of chirality relates to single molecules. This article deals with cases in which exciton chirality is generated by the interaction of associated carotenoids. The handed property responsible for exciton signals in these systems is due to the alignment of neighboring molecules held together by secondary chemical forces. Their mutual positions are characterized by the overlay angle. Experimental manifestation is obtained by spectroscopic studies on carotenoid aggregates. Compared to molecular spectra, both UV/visible and circular dichroism spectroscopic observations reveal modified absorption bands and induced Cotton effects of opposite sign (exciton couplets), respectively. A new term, "supramolecular exciton chirality," is suggested for these phenomena, allowing the detection of weak chemical interactions not readily accessible for experimental studies, although highly important in the mechanism of biological processes.     

Characterisation of the progression of azaserine-induced rat pancreatic adenocarcinoma by proliferative cell nuclear antigen,basement membrane laminin and trypsinogen immunohistochemistry

The progression of azaserine-induced rat pancreatic adenocarcinoma (AC) was characterised using quantitative and semiquantitative immunohistochemistry for proliferating cell nuclear antigen (PCNA), basement membrane laminin (BML) and trypsinogen (TG). Samples were taken 5-20 months after initiation. High PCNA-labelling indices (PCNA LIs) were measured 5 months after the induction of atypical acinar cell nodules (AACNs), which decreased later and stagnated until a further decline in the month 10 adenomas. Then a second premalignant proliferative wave was observed (month 13) within the adenoma stage. Later, in month 20 differentiated ACs PCNA LIs fell to the host tissue level but were found highest in the month 20 anaplastic ACs indicating a switch to malignant proliferation. Month 20 invasive ACs showed a number of separate proliferative foci. In early AACNs, BML decreased and remained low till the local maximum in the month 13 adenoma. Invasive ACs did not express BML. Month 5 AACN and differentiated AC were TG deficient but anaplastic AC regained its TG expression. However invasive AC was again TG negative. These results are discussed in combination with our previous data on progressional changes of autophagic capacity and microvessel densities.     

ABA treatment increases both the desiccation tolerance of photosynthesis,and nonphotochemical quenching in the moss Atrichum undulatum

Pulse amplitude modulation fluorescence was used to investigate whether abscisic acid (ABA) pretreatment increases the desiccation tolerance of photosynthesis in the moss Atrichum undulatum. In unstressed plants, ABA pretreatment decreased the F _V/F _m ratio, largely as a result of an increase in F _o. This indicated a reduction in energy transfer between LHCII and PSII, possibly hardening the moss to subsequent stress by reducing the production of the reactive oxygen species near PSII. During desiccation, F ₀, F _m, F _v/F _m, PSII, and NPQ and F ₀ quenching declined in ABA-treated and nontreated mosses. However, during rehydration, F ₀, F _m, F _v/F _m, and PSII recovered faster in ABA-treated plants, suggesting that ABA improved the tolerance of photosystem II to desiccation. NPQ increased upon rehydration in mosses from both treatments, but much more rapidly in ABA-treated plants; during the first hour of rehydration, NPQ was two-fold greater in plants treated with ABA. F ₀quenching followed a similar pattern, indicating that ABA treatment stimulated zeaxanthin-based quenching. The implications of these results for the mechanisms of ABA-induced desiccation tolerance in A. undulatum are discussed.     

Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid‐transfer proteins encoded by the barley low‐temperature‐inducible gene family, blt4

The homology modelling technique was used to predict the tertiary structures of three members of the low-temperature-inducible barley vegetative shoot epidermal lipid-transfer protein (LTP) family, BLT4, on the basis of the X-ray crystallographically determined three-dimensional structure of a maize seedling LTP. Differences between the maize LTP and the BLT4 family include amino acid substitutions around the entrance and inside the predicted hydrophobic binding tunnels of these proteins. Because of the deletion of the loop region corresponding to Val60–Gly62 of the maize LTP from all three BLT4 LTPs, their internal hydrophobic tunnels are longer. Molecular dynamics modelling shows that BLT4.9 can accommodate hexadecanoic acid in its binding tunnel in similar conformation to the maize LTP. However, modelled cis,cis-9,12-octadecandienoic acid had a more favourable interaction with the BLT4.9 LTP than with the maize protein. Di-cis,cis-9,12-octadecandienoyl phosphatidylglycerol and di-cis,cis-9,12-octadecandienoyl phosphatidylcholine were modelled in the BLT4.9 structure with the fatty acyl group at position 1 embedded in the binding tunnel and the group at position 2 located on the solvent accessible surface of the protein. The results of the modelling suggest that the phospholipid headgroup can form hydrogen and salt bridges with polar and charged residues outside the binding tunnel and the exposed hydrocarbon chain interacts with hydrophobic amino acids on the surface. These results are consistent with the proposal that BLT4 LTPs have a lipid-transfer function associated with frost acclimation in barley.     

Study of the effect of lactose on the structure of sodium alginate films

The aim of this study was to evaluate the interaction between the film-forming sodium alginate and lactose monohydrate. This combination is used in the co-spray-drying technique for microencapsulation, but no respect on the structure of the film formed has not been published previously. From mechanical tests, positronium lifetime measurements and FT-IR studies on free films containing different ratios of film-former and lactose, we concluded that the mechanical strength of the sodium alginate film decreased with the increasing proportion of lactose. The free volume in the polymer matrix decreased to a minimum as the lactose content was progressively increased to 40%, but subsequently increased at higher lactose contents. The explanation of this phenomenon is the filling of the holes with the sugar. As lactose became predominant component, the structure of the polymer network weakened. These conclusions were supported by the FT-IR findings. The present results permit a clear explanation of the previously reported favourable effects of this film-forming combination on the dissolution of the active agent from the microcapsules.     

Cool Headed Individuals Are Better Survivors: Non-Consumptive and Consumptive Effects of a Generalist Predator on a Sap Feeding Insect

Non-consumptive effects (NCEs) of predators are part of the complex interactions among insect natural enemies and prey. NCEs have been shown to significantly affect prey foraging and feeding. Leafhopper''s (Auchenorrhyncha) lengthy phloem feeding bouts may play a role in pathogen transmission in vector species and also exposes them to predation risk. However, NCEs on leafhoppers have been scarcely studied, and we lack basic information about how anti-predator behaviour influences foraging and feeding in these species. Here we report a study on non-consumptive and consumptive predator-prey interactions in a naturally co-occurring spider–leafhopper system. In mesocosm arenas we studied movement patterns during foraging and feeding of the leafhopper Psammotettix alienus in the presence of the spider predator Tibellus oblongus. Leafhoppers delayed feeding and fed much less often when the spider was present. Foraging movement pattern changed under predation risk: movements became more frequent and brief. There was considerable individual variation in foraging movement activity. Those individuals that increased movement activity in the presence of predators exposed themselves to higher predation risk. However, surviving individuals exhibited a ‘cool headed’ reaction to spider presence by moving less than leafhoppers in control trials. No leafhoppers were preyed upon while feeding. We consider delayed feeding as a “paradoxical” antipredator tactic, since it is not necessarily an optimal strategy against a sit-and-wait generalist predator.