Cryptochrome Mediates Light-Dependent Magnetosensitivity of Drosophila's Circadian Clock

Since 1960, magnetic fields have been discussed as Zeitgebers for circadian clocks, but the mechanism by which clocks perceive and process magnetic information has remained unknown. Recently, the radical-pair model involving light-activated photoreceptors as magnetic field sensors has gained considerable support, and the blue-light photoreceptor cryptochrome (CRY) has been proposed as a suitable molecule to mediate such magnetosensitivity. Since CRY is expressed in the circadian clock neurons and acts as a critical photoreceptor of Drosophila's clock, we aimed to test the role of CRY in magnetosensitivity of the circadian clock. In response to light, CRY causes slowing of the clock, ultimately leading to arrhythmic behavior. We expected that in the presence of applied magnetic fields, the impact of CRY on clock rhythmicity should be altered. Furthermore, according to the radical-pair hypothesis this response should be dependent on wavelength and on the field strength applied. We tested the effect of applied static magnetic fields on the circadian clock and found that flies exposed to these fields indeed showed enhanced slowing of clock rhythms. This effect was maximal at 300 μT, and reduced at both higher and lower field strengths. Clock response to magnetic fields was present in blue light, but absent under red-light illumination, which does not activate CRY. Furthermore, cry^b and cry^OUT mutants did not show any response, and flies overexpressing CRY in the clock neurons exhibited an enhanced response to the field. We conclude that Drosophila's circadian clock is sensitive to magnetic fields and that this sensitivity depends on light activation of CRY and on the applied field strength, consistent with the radical pair mechanism. CRY is widespread throughout biological systems and has been suggested as receptor for magnetic compass orientation in migratory birds. The present data establish the circadian clock of Drosophila as a model system for CRY-dependent magnetic sensitivity. Furthermore, given that CRY occurs in multiple tissues of Drosophila, including those potentially implicated in fly orientation, future studies may yield insights that could be applicable to the magnetic compass of migratory birds and even to potential magnetic field effects in humans.     

Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.     

Immunohistochemical study of N-epsilon-carboxymethyl lysine (CML) in human brain: relation to vascular dementia

Background  

Advanced glycation end-products (AGEs) and their receptor (RAGE) occur in dementia of the Alzheimer's type and diabetic microvascular disease. Accumulation of AGEs relates to risk factors for vascular dementia with ageing, including hypertension and diabetes. Cognitive dysfunction in vascular dementia may relate to microvascular disease resembling that in diabetes. We tested if, among people with cerebrovascular disease, (1) those with dementia have higher levels of neuronal and vascular AGEs and (2) if cognitive dysfunction depends on neuronal and/or vascular AGE levels.     

δ(13) C of leaf-respired CO(2) reflects intrinsic water-use efficiency in barley

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley.     

The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.     

Tissue section AFM: In situ ultrastructural imaging of native biomolecules

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.     

Par-4 links dopamine signaling and depression

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4DeltaLZ. Primary neurons from Par-4DeltaLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4DeltaLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.     

Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1

We recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind specifically to the surface glycoprotein (gp 120) of the R5 strain, HIV-1(Ba-L), as presented in a previous study. These aptamers potently neutralize HIV-1 infectivity in human peripheral blood mononuclear cells of both tissue culture lab-adapted strains and diverse R5 clinical isolates from multiple clades. Here, we report a detailed structural characterization of one such neutralizing aptamer, B40, using enzymatic and chemical probing methods. We identify the minimal region of the aptamer essential for binding gp120 and accordingly design a 77-nucleotide truncated aptamer, B40t77. We then quantitatively analyze the binding affinity and neutralization potency of the parental and truncated (minimal) aptamer, and show them to be comparable. Furthermore, using results from secondary structure analysis, RNA mutagenesis and BIAcore surface plasmon resonance (SPR) binding assays, we hypothesize that a folded RNA structure is required to present specific nucleotide sequences to allow gp120-recognition and binding. The information gained from this study may provide leads for development of novel anti-HIV-1 therapies and can be used to extend our understanding of the molecular interactions between the virus and its host cell.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.