Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.     

Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless(1) (ecd(1)) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd(1) conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd(-) clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.     

Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. We provide evidence that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. We show that the initial membrane association of Dgo depends on Fz, and that Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi.     

Comparison of terpene composition in Engelmann spruce (Picea engelmannii) using hydrodistillation, SPME and PLE

Terpenes emitted by conifer trees are generally determined by analysing plant extracts or essential oils, prepared from foliage and cones using steam distillation. The application of these procedures limits experiments to cut plant materials. Recently headspace techniques have been adopted to examine terpene emission by living plants. This paper deals with the application of solid-phase micro-extraction (SPME) for the analysis of terpenes emitted by conifers foliage of Engelmann spruce (Picea engelmannii), including its seedlings. The compositions of SPME extracts obtained for destroyed and non-destroyed old and juvenile spruce needles were compared with the compositions of essential oils and pressurised liquid extraction (PLE) extracts corresponding to the same plant materials. No substantial differences have been found in the qualitative terpene composition estimated by analysing essential oil and PLE and SPME extracts from non-destroyed old and juvenile foliage. The disintegration of spruce needles results in the formation of a significant amount of myrcene in the case of the old conifer foliage and non-terpenoic compounds in the case of juvenile conifer foliage. This phenomenon can be attributed to enzymatic reactions occurring in the destroyed plant cells.     

Application of oil refinery waste in the biosynthesis of glycolipids by yeast

Candida antarctica or Candida apicola synthesized surfactants (glycolipids) in the cultivation medium supplemented with oil refinery waste, either with soapstock (from 5.0% to 12.0% v/v) or post-refinery fatty acids (from 2.0% to 5.0% v/v). The efficiency of glycolipids synthesis was determined by the amount of waste supplemented to the medium and was from 7.3 to 13.4 g/l and from 6.6 to 10.5 g/l in the medium supplemented with soapstock and post-refinery fatty acids, respectively. The studied yeast also synthesized glycolipids in the non-supplemented medium however, by the enrichment of medium with the oil refinery waste, a 7.5-8.5-fold greater concentration of glycolipids was obtained in the post-culture liquid then in the medium without addition of oil refinery waste. The yeast synthesized from 6.6 to 10.3 g dry biomass/l and the intra-cellular fat content was from 16.8% to 30.2%. The efficiency of glycolipids synthesis was determined by yeast species, medium acidity and culture period. The surface tension of the post-culture liquid separated from yeast biomass was reduced to 35.6 mN/m, which corresponded to the surface tension obtained at the critical micelle concentration of glycolipids.     

Tolerance induced by the lipopeptide Pam3Cys is due to ablation of IL-1R-associated kinase-1

Stimulation of the human monocytic cell line Mono Mac 6 with the synthetic lipopeptide (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys(4)-OH, trihydrochloride (Pam(3)Cys) at 10 microg/ml induces a rapid expression of the TNF gene in a TLR2-dependent fashion. Preculture of the cells with Pam(3)Cys at 1 microg/ml leads to a reduced response after subsequent stimulation with Pam(3)Cys at 10 microg/ml, indicating that the cells have become tolerant to Pam(3)Cys. The CD14 and TLR2 expression is not decreased on the surface of the tolerant cells, but rather up-regulated. Analysis of the NF-kappaB binding in Pam(3)Cys-tolerant cells shows a failure to mobilize NF-kappaB-p50p65 heterodimers, while NF-kappaB-p50p50 homodimers remain unchanged. Pam(3)Cys-tolerant cells showed neither IkappaBalpha-Ser(32) phosphorylation nor IkappaBalpha degradation but MyD88 protein was unaltered. However, IRAK-1 protein was absent in Pam(3)Cys-induced tolerance, while IRAK-1 mRNA was still detectable at 30% compared with untreated cells. In contrast, in LPS-tolerized cells, p50p50 homodimers were induced, IRAK-1 protein level was only partially decreased, and p50p65 mobilization remained intact. It is concluded that in Mono Mac 6 monocytic cells, inhibition of IRAK-1 expression at the mRNA and protein levels is the main TLR-2-dependent mechanism responsible for Pam(3)Cys-induced tolerance, but not for TLR-4-dependent LPS-induced tolerance.     

RAPD-PCR analysis of various goose populations

The aim of this study was to genetically analyse by the RAPD-PCR method four indigenous Polish goose breeds, Kartuska (Ka), Lubelska (Lu), Kielecka (Ki) and Podkarpacka (Pd), in order to determine the band-sharing frequency as well as bands characteristic of the evaluated breeds. The birds were maintained as conservative flocks, accounting for a reserve of genetic resources. A total of 102 scorable bands were obtained, their number ranging from 0 to 8, depending on one of seven primers used and the group of birds analyzed, within a mean of 3.64. For each genetic group specific bands with given primers were obtained, suggesting their potential for use as population-specific markers, especially in ex-situ conservation methods. The results also suggest that keeping endangered geese as separate flocks is relevant for their preservation.     

Assessment of the effect of alpha-galactosides injected during embryogenesis on selected chicken traits

The effect of different doses of alpha-galactoside (RFOs) preparations from Pisum sativum L. cv. Opal, injected into eggs during embryogenesis, on maintaining a high number of bifidobacteria, selected chicken broiler traits and the lipoprotein level of blood were studied. Two independent experiments were conducted. In the first, Ringer water solution containing 1.763 mg/egg of fructooligosaccharides (FOS) (I group), 2.1158 mg of pea RFO preparation containing 20% sucrose (II group) and 0.4232 mg of sucrose (III group) were injected into Hubbard broiler breeder eggs containing 12-day old embryos. Only Ringer water solution was applied to the eggs of the control group (IV group). The number of bifidobacteria determined in faeces of two-day old chicken of groups I and II was significantly higher in comparison with the sucrose and control groups. The high level ofbifidobacteria of groups I and II was maintained during 6 weeks. The dose of both preparations had no influence on the body weight, carcass, breast muscle, leg and abdominal fat ratio, total cholesterol, HDL and LDL serum concentrations. Broiler mortality and breast muscle cholesterol concentration was highest (P < 0.05) for the control group. On the other hand, the European Production Index, as well as serum triglycerides, were the lowest for this group. The second experiment was performed on Hybro G chicken breeder eggs. 0.69, 3.43 and 6.87 mg/egg of pea RFO preparation doses containing 20% sucrose were injected into the experimental groups. The number of bifidobacteria in the caecum and selected meat traits of broilers were determined. The results of this experiment confirmed that RFO injection in ovo causes the long-time maintenance of a high level ofbifidobacteria. The dose of the preparations does not have any effect on the selected broiler meat traits, except that the highest dose increases the percent of carcase in body weight. However, this dose reduced the hatchability of the treated embryos.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.