Single-minded and the evolution of the ventral midline in arthropods

Glypicans are multifunctional proteoglycans with regulatory roles in several intercellular signaling pathways. Here, we examine the functional requirements for glypican regulation of bone morphogenetic protein (BMP)-mediated body length in C. elegans. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit BMP signaling in vivo: the N-terminal furin protease product and the C-terminal region containing heparan sulfate attachment sequences. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. Cleavage of LON-2 into two parts at the conserved furin protease site is not required for LON-2 to inhibit BMP-like signaling. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 activity. Finally, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity, suggesting that LON-2 inhibits BMP signaling by acting as a scaffold for BMP and an RGD-binding protein.     

Novel method to quantify neuropil threads in brains from elders with or without cognitive impairment.

Pathological alterations in dendrites and axons (i.e., neuritic pathologies) occur in the normal aging brain as well as in brains from elders with mild cognitive impairment and neurodegenerative dementia. These alterations may correlate with clinical measures of cognitive abilities, but the contribution of neuropil threads (NTs), which constitute 85-90% of cortical tau pathology, has not been clear because of the lack of quantitative methodologies. We combined quantitative fractionation and image analysis to devise a strategy for measuring the burden of tau-rich NTs in the entorhinal and perirhinal cortex of brains from elders with and without cognitive impairment, including dementia due to Alzheimer's disease (AD). On the basis of data presented here using this novel strategy, we conclude that this quantitative imaging technique will facilitate efforts to determine the behavioral correlations of neuritic lesions in AD and other brain disorders.     

Delayed onset of midline netrin expression in Artemia franciscana coincides with commissural axon growth and provides evidence for homology of midline cells in distantly related arthropods

Although many similarities in arthropod central nervous systems (CNS) development exist, differences in midline cell formation and ventral nerve cord axonogenesis have been noted in arthropods. It is possible that changes in the expression of axon guidance molecules such as Netrin, which functions during commissural axon guidance in Drosophila and many other organisms, may parallel these differences. In this investigation, we analyze this hypothesis by examining Netrin accumulation during development of the brine shrimp Artemia franciscana, a branchiopod crustacean. An Artemia franciscana netrin (afrnet) orthologue was cloned. An antibody to the afrNet protein was generated and used to examine the pattern of afrNet accumulation during Artemia development. Despite differences between Drosophila and Artemia nerve cord development, examination of afrNet accumulation suggests that this protein functions to regulate commissure formation during Artemia CNS development. However, detection of afrNet at the midline and on commissural axons occurs at a relatively later time point in Artemia as compared with Drosophila. Detection of afrNet in a subset of midline cells that closely resemble Netrin-expressing cells at the Drosophila midline provides evidence for homology of midline cells in arthropods. Expression of Netrins in many other tissues is comparable, suggesting that Netrin proteins may play many conserved roles during arthropod development.     

Identification of common and unique modifiers of zebrafish midline bifurcation and cyclopia

Loss of the zebrafish Nodal-related protein Squint causes a spectrum of phenotypes including cyclopia and midline bifurcations (MB). Here we examine MBs and their relation to cyclopia in maternal-zygotic squint (MZsqt) mutants. There is a concordance of MB with cyclopia in MZsqt embryos. Heat treatment and depletion of Hsp90a are “common” risk factors, each of which increases the incidence of both phenotypes. Midline identity is specified on both sides of MBs, and deep-layer cells are initially lacking within bifurcations, whereas enveloping layer cells are intact. Bifurcations do not appear until the completion of gastrulation and are preceded by gaps in the expression of wnt5b, an essential regulator of dorsal convergence. The incidence of early MBs and wnt5b expression defects in heated MZsqt embryos is high, but there is also substantial recovery. Wnt5b depletion increases the incidence of MB, but not cyclopia, and as such Wnt5b is a “unique” risk factor for MB. Reciprocally, depletion of Wnt11 or Hsp90b increases cyclopia only. In summary, we find that MB arises after gastrulation in regions that fail to express wnt5b, and we show that two complex dysmorphologies - MB and cyclopia - can be promoted by either common or unique risk factors.     

ATPases: Common and unique features within a group of enzymes

An attempt is made to survey ATPases with respect to features common to all or some of them and features peculiar to each individual enzyme of the group. Clues are presented for a tentative classification of ATPases and a simple system is suggested for the designation of interaction of ATPases with ions which is often used as the main feature for identification of individual ATPases.     

Distribution of extracellular glutamate in the neuropil of hippocampus

Reported values of extracellular glutamate concentrations in the resting state depend on the method of measurement and vary ～1000-fold. As glutamate levels in the micromolar range can cause receptor desensitization and excitotoxicity, and thus affect neuronal excitability, an accurate determination of ambient glutamate is important. Part of the variability of previous measurements may have resulted from the sampling of glutamate in different extracellular compartments, e.g., synaptic versus extrasynaptic volumes. A steep concentration gradient of glutamate between these two compartments could be maintained, for example, by high densities of glutamate transporters arrayed at the edges of synapses. We have used two photon laser scanning microscopy and electrophysiology to investigate whether extracellular glutamate is compartmentalized in acute hippocampal slices. Pharmacological blockade of NMDARs had no effect on Ca(2+) transients generated in dendritic shafts or spines of CA1 pyramidal neurons by depolarization, suggesting that ambient glutamate is too low to activate a significant number of NMDARs. Furthermore, blockade of transporters did not flood the synapse with glutamate, indicating that synaptic NMDARs are not protected from high concentrations of extrasynaptic glutamate. We suggest that, in the CA1 region of hippocampus, glutamate transporters do not create a privileged space within the synapse but rather keep ambient glutamate at very low levels throughout the neuropil.     

A non-intuitive design of a cyclic decapeptide library with unique backbone structural features

An analysis of hydrogen bonding patterns of cyclic decapeptide (CDP) beta-sheet structures has resulted in a 'non-intuitive' design of cyclic decapeptides wherein their beta-turns and residue positions can be fixed by choosing 2 of the 10 residues, i.e. positions i and i+4, to be Prolines or N-substituted residues. This sequence relationship between the two Pro or N-substituted residues is shown to uniquely define the conformation of the CDP. Furthermore, this design of the 2 beta-turn, beta-sheet CDP structure is expected to be characterised by residues disposed in an exclusive fashion in which four residues are on one side of the ring, two on the other and the four corner residues in the beta-turn are in the plane of the ring. This opens up the possibility of fine-tuning the four residues facing one way and /or the two residues facing the other way such that a library containing a myriad of chemically diverse systems could be obtained. The design process along with the molecular modelling of specific CDP-s and the building of a CDP library are discussed in detail.     

Of blood, brains and bacteria, the Amt/Rh transporter family: emerging role of Amt as a unique microbial sensor

Members of the Amt/Rh family of transporters are found almost ubiquitously in all forms of life. However, the molecular state of the substrate (NH₃ or NH₄⁺) has been the subject of active debate. At least for bacterial Amt proteins, the model emerging from computational, X-ray crystal and mutational analysis is that NH₄⁺ is deprotonated at the exterior, conducted through the membrane as NH_3, and reprotonated at the cytoplasmic interface. A proton concomitantly is transferred from the exterior to the interior, although the mechanism is unclear. Here we discuss recent evidence indicating that an important function of at least some eukaryotic and bacterial Amts is to act as ammonium sensors and regulate cellular metabolism in response to changes in external ammonium concentrations. This is now well documented in the regulation of yeast pseudohyphal development and filamentous growth. As well, membrane sequestration of GlnK, a PII signal transduction protein, by AmtB has been shown to regulate nitrogenase in some diazotrophs, and nitrogen metabolism in some Gram-positive bacteria. Formation of GlnK–AmtB membrane complexes might have other, as yet undiscovered, regulatory roles. This possibility is emphasized by the discovery in some genomes of genes for chimeric Amts with fusions to various regulatory elements.     

Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis

Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.     

昆虫脑神经髓结构图谱构建方法综述

脑控制和调节所有动物的行为,昆虫也不例外,构建脑神经髓结构图谱有利于阐明其对行为调控的神经机制.目前,除一些模式昆虫的脑图谱被构建外,大多数昆虫仅针对少数易识别的神经髓(如视叶、触角叶和蕈形体等)进行了三维重建,而对脑内大部分区域还未描述,这与其结构的复杂性有关.随着共聚焦显微成像和计算机三维重建技术的发展,人们有机会...