The role of survivin in angiogenesis during zebrafish embryonic development

Background  

Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (Sur_UTR) and sequences flanking the initiation codon (Sur_ATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP) ^y1 embryos. Results: In embryos co-injected with Sur_UTR and Sur_ATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of Sur_UTR and Sur_ATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.     

New species of the snakefly genus Mongoloraphidia (Raphidioptera: Raphidiidae) from Japan and Taiwan,with phylogenetic and biogeographical remarks on the Raphidiidae of Eastern Asia

Two species of the snakefly genus Mongoloraphidia Aspöck & Aspöck, 1968 from Japan and Taiwan are described as new to science: Mongoloraphidia (Japanoraphidia) occidentalis sp. nov. and Mongoloraphidia (Formosoraphidia) curvata sp. nov. A key to the species of Mongoloraphidia from Eastern Asia is provided. Phylogenetic and biogeographical aspects on the Raphidiidae from Eastern Asia are discussed.     

Inhibition of dopamine beta-hydroxylase by thiazoline-2-carboxylate, a suspected physiological product of D-amino acid oxidase

Thiazoline-2-carboxylate was chemically synthesized and shown to be identical in all respects to the product formed in a D-amino acid oxidase catalyzed reaction involving cysteamine and glyoxylate. Both the chemically synthesized and enzymically prepared thiazoline-2-carboxylate are effective inhibitors of dopamine β-hydroxylase but they do not appreciably affect the activity of several other metalloenzymes that require copper, iron or zinc. The inhibition of dopamine β-hydroxylase is competitive with respect to the reactant ascorbic acid and uncompetitive with respect to tyramine. The possible physiological significance of this inhibition is briefly considered.     

Zinc exchange by blood cells in nearly physiologic standard conditions

Determination of zinc concentrations in white blood cells has been used to establish zinc deficiency. During pathological conditions changes in zinc concentrations in these blood cells were observed. However, these investigations were hampered by the low amount of zinc in this form per mL blood. Earlier we demonstrated that, in the case of zinc deficiency, the uptake of zinc was increased, using the in vitro exchange of zinc by the various blood cells with extracellular zinc labeled with⁶⁵Zn in fairly physiologic conditions. In case of inflammation, no increase in zinc uptake by erythrocytes was seen, indicating that this method probably can be used to differentiate real from apparent zinc deficiency. Only during the first days of the inflammatory process, probably representing the redistribution phase during which zinc moves from the serum to the liver, a small increase in in vitro zinc uptake was seen in mononuclear cells (MNC) and polymorphonuclear cells (PMNC). Earlier papers raised some questions; e.g., is the uptake part of an exchange process and can the efflux of zinc by the cells be measured by the same method; what is the influence of time on the process of zinc uptake; what is the magnitude of the uptake of zinc by the cells compared to the zinc concentration in the cells; and, what is the influence of temperature on the uptake of zinc? In the present study, the influence of incubation time and temperature on the uptake of zinc by human and rat blood cells and on the release of zinc by rat blood cells was studied. At least three phases of uptake of zinc in the various cells were found by varying the incubation time—a fast phase during the first half hour, probably caused by an aspecific binding of zinc on or in the cell membrane; a second fast uptake between 60–330 min, probably caused by an influx of zinc in the cell as part of the exchange process of zinc; and a slow third phase after 5.5 h, in which probably the in- and efflux of the rapidly exchangeable intracellular pool is more or less equilibrated. For mononuclear cells, polymorphonuclear cells, and erythrocytes of rats, the rapidly exchangeable intracellular pool is 40%, 53%, and 10%, respectively, of the total zinc content of the cells. This study is also performed in human cells; in human cells the exchangeable pool of mononuclear cells and erythrocytes is 17 and 3.5% of the total zinc content of the cells, respectively. The efflux of zinc by blood cells can be measured by the same method. Both the uptake and the loss of zinc by blood cells of rats were compared and are of the same magnitude, indicating that the in vitro uptake of zinc described elsewhere is part of an exchange process. Increasing temperature during incubation procedures results in an increase of zinc uptake by human blood cells, even at high temperatures of 41°C, although there are gradual differences between the various blood cells. Both the in- and efflux of zinc by blood cells are very small at 4°C.     

Patch size effects are more important than genetic diversity for plant–herbivore interactions in Brassica crops

1. Considerable evidence suggests that the diversity within plant communities may strongly affect the strength of species interactions, but the majority of studies only considered interspecific diversity. 2. This paper examines the effect of intraspecific genetic diversity within Brassica fields on two Brassica specialists, cabbage root fly, and diamondback moth, and on a parasitoid attacking diamondback moths. Genetic diversity was manipulated both in a replacement and an additive design. 3. Both herbivore densities and parasitism rates were higher in smaller plots, with limited responses to increased within‐plot diversity. All species showed variable densities across genotypes, and preference hierarchies were species specific. 4. Responses to plot size in root flies scaled with the diameter‐to‐area ratio, suggesting that patch detectability affected local density, whereas responses by diamondback moths and parasitoids deviated from this ratio. These species differences could be traced to differences in the residence time within patches, where diamondback moths typically spend longer and more variable time periods in patches than root flies. 5. The lack of response to genetic diversity by both herbivores suggests that egg‐laying rates are affected by decisions on the plant and not by attraction from a distance, neither to the plant itself nor the patch. Patterns of differential attack may then be due to different acceptability for studied genotypes. 6. Future theories on insect responses to spatial heterogeneity should focus on species traits and how traits interact with information landscapes in the field.     

Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy

Crystal structures of the molecular motor kinesin show conformational variability in a structural element called the neck linker. Conformational change in the neck linker, initiated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and 'docked'). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker conformation, which we confirmed by solving a new crystal structure. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility.     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Prompt inhibition of fMLP-induced Ca2+ mobilization by parenteral lipid emulsions in human neutrophils

It remains unclear whether modulation of immune system functions by lipids contributes to the increased infection rate observed in patients treated with parenteral nutrition. We therefore evaluated the effects of lipid emulsions derived from fish oil [very long chain triglycerides (VLCT)], olive oil [long-chain triglycerides- mono-unsaturated fatty acid (LCT-MUFA)], soya oil [long-chain triglycerides (LCT)], or a physical mixture of coconut and soya oil [mixed long- and medium-chain triglycerides (LCT-MCT)] on neutrophil activation. N-formyl-methionyl-leucyl-phenylalanine (fMLP) evoked an immediate increase of the cytosolic Ca2+ concentration ([Ca2+](i,av)) in a suspension of neutrophils. When added 3 min before fMLP, however, all four lipid emulsions reduced the hormone-induced increase in [Ca2+](i,av) with the same efficacy but with different potency. Half-maximal inhibition was reached at emulsion concentrations of 0.24 mM VLCT, 0.32 mM LCT-MCT, 0.52 mM LCT, and 0.82 mM LCT-MUFA. Similarly to the lipids, the protein kinase C (PKC) activator PMA markedly reduced the fMLP-induced increase in [Ca2+](i,av). PMA inhibition was abolished by the PKC inhibitor staurosporine. In contrast, however, this drug did not interfere with the inhibitory lipid effect, indicating that the lipids act primarily in a PKC-independent manner.In summary, this study shows that nutritional lipids can evoke a prompt and significant attenuation of hormone-induced neutrophil stimulation and that the emulsions based on fish oil and a mixture of coconut oil and soya oil are among the most potent ones in this respect.