Endocannabinoids mediate neuron-astrocyte communication

Cannabinoid receptors play key roles in brain function, and cannabinoid effects in brain physiology and drug-related behavior are thought to be mediated by receptors present in neurons. Neuron-astrocyte communication relies on the expression by astrocytes of neurotransmitter receptors. Yet, the expression of cannabinoid receptors by astrocytes in situ and their involvement in the neuron-astrocyte communication remain largely unknown. We show that hippocampal astrocytes express CB1 receptors that upon activation lead to phospholipase C-dependent Ca2+ mobilization from internal stores. These receptors are activated by endocannabinoids released by neurons, increasing astrocyte Ca2+ levels, which stimulate glutamate release that activates NMDA receptors in pyramidal neurons. These results demonstrate the existence of endocannabinoid-mediated neuron-astrocyte communication, revealing that astrocytes are targets of cannabinoids and might therefore participate in the physiology of cannabinoid-related addiction. They also reveal the existence of an endocannabinoid-glutamate signaling pathway where astrocytes serve as a bridge for nonsynaptic interneuronal communication.     

Detection and Structural Characterization of Ether Glycerophosphoethanolamine from Cortical Lysosomes Following Traumatic Brain Injury Using UPLC‐HDMSE

The use of ultra performance liquid chromatography coupled to data independent tandem mass spectrometry with traveling wave ion mobility for detection and structural identification of ether‐linked glycerophosphoethanolamine is described. The experimental design generates 4D data (chromatographic retention time, precursor accurate mass, drift time with associated calculated collisional cross‐section, and time‐aligned accurate mass diagnostic product ions) for each ionization mode. Confident structure identification depends on satisfying 4D data confirmation in both positive and negative ion mode. Using this methodology, a number of ether‐linked glycerophosphoethanolamine lipids are structurally elucidated from mouse brain lysosomes. It is further determined that several ether‐linked glycerophosphoethanolamine structures are differentially abundant between lysosomes isolated from mouse cortex following traumatic brain injury as compared to that of sham animals. The combined effort of aligning multi‐dimensional mass spectrometry data with a well‐defined traumatic brain injury model lays the foundation for gaining mechanistic insight in the role lysosomal membrane damage plays in neuronal cell death following brain injury.     

Genistein effects on growth and cell cycle ofCandida albicans

Microbial virulence is generally considered to be multifactorial with infection resulting from the sum of several globally regulated virulence factors. Estrogen may serve as a signal for global virulence induction in Candida albicans. Nonsteroidal estrogens and estrogen receptor antagonists may therefore have interesting effects on yeast and their virulence factors. Growth of C. albicans was monitored by viable plate counts at timed intervals after inoculation into yeast nitrogen broth plus glucose. To determine if increased growth of yeast in the presence of estradiol was due to tyrosine kinase-mediated signaling, we measured growth in the presence of genistein, estradiol or genistein plus estradiol and compared these conditions to controls, which were not supplemented with either compound. Unexpectedly, genistein stimulated growth of C. albicans. In addition, genistein was found to increase the rate of germination (possibly reflecting release from G(0) into G(1) cell cycle phase) and also increased Hsp90 expression, demonstrated by a dot blot technique which employed a commercial primary antibody detected with chemiluminescence with horseradish peroxidase-labeled secondary antibody. These biological effects may be attributable to genistein's activity as a phytoestrogen. In contrast, nafoxidine suppressed growth of Candida and mildly diminished Hsp90 expression. This study raises the possibility of receptor cross-talk between estrogen and isoflavinoid compounds, and antiestrogens which may affect the same signaling system, though separate targets for each compound were not ruled out.     

The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

Cyclic N⁶-threonylcarbamoyladenosine (‘cyclic t⁶A’, ct⁶A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct⁶A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct⁶A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t⁶A to form ct⁶A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K⁺ and Na⁺ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t⁶A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct⁶A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preserving avian blood and DNA sampled in the wild: A survey of personal experiences

Collecting and storing biological material from wild animals in a way that does not deteriorate DNA quality for subsequent analyses is instrumental for research in ecology and evolution. Our aims were to gather reports on the effectiveness of methods commonly used by researchers for the field collection and long‐term storage of blood samples and DNA extracts from wild birds. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses but were not related to season of blood sampling, access to equipment, transporting samples, temperature, and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.