A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. We have now identified alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. Our data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells.     

Studies on p53 and Bax protein expression in Cockayne syndrome cells after UV irradiation and interferon-beta treatment

Human interferon (HuIFN) has a protective effect against ultraviolet (UV)-induced killing of Cockayne syndrome (CS) and xeroderma pigmentosum (XP) cells. Irradiation with ultraviolet (UV) resulted in nuclear accumulation of p53 in normal human fibroblast cells, and this accumulation was suppressed by treatment with HuIFN-beta. On the other hand, a large amount of p53 was found in both nuclear and cytoplasmic fractions of one SV40-transformed XP and two SV40-transformed CS cell strains irrespective of UV irradiation. Treatment with HuIFN-beta reduced the level of pro-apoptotic Bax protein without suppression of nuclear accumulation of p53 in the CS cells but not in the XP cells. These findings suggest that there are different mechanisms of UV-refractoriness caused by HuIFN-beta in UV-sensitive CS and XP cells.     

Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.     

Acute toxicity of neonicotinoids and some insecticides to first instar nymphs of a non-target damselfly, <Emphasis Type="Italic">Ischnura senegalensis</Emphasis> (Odonata: Coenagrionidae), in Japanese paddy fields

Systemic insecticides such as neonicotinoids and fipronil are widely applied in rice production. These insecticides have been suspected of reducing biodiversity in paddy ecosystems and reducing wild dragonfly populations in Japan. Conventional ecotoxicological risk assessment could not confirm this, as it has not considered interspecific variation in sensitivity to insecticides. We estimated the median effect concentration (EC50) of 15 systemic insecticides to first instar nymphs of a Japanese damselfly, Ischnura senegalensis (Rambur) (Odonata: Coenagrionidae), commonly found in rice paddy fields. Damselflies were found to be highly sensitive to pyrethroid pesticides, less so to phenylpyrazole, organophosphates, and carbamates, and least sensitive to neonicotinoids, nereistoxin, and diamide. Given the acute toxicity data, the sensitivity of the damselfly to neonicotinoids was considered to be lower than that of other aquatic insects, whereas the EC50 values of the damselfly were 2–3 orders lower than that of Daphnia magna Straus (Diplostraca: Daphniidae), which is a standard test species. These results indicate that the conventional ecological risk assessment based on acute toxicity data of D. magna would underestimate the impact of neonicotinoids on Odonata diversity in paddy ecosystems. We therefore recommend using the paddy-dwelling damselfly as a new test species for insecticide bioassay.     

Local distribution of collagen fibers determines crack initiation site and its propagation direction during aortic rupture

Although elucidation of the mechanism of aortic aneurysm rupture is important, the characteristics of crack initiation and propagation sites remain unknown. To determine the microscopic properties of these sites, the characteristics of local strains and constituents at crack initiation and propagation sites were investigated during biaxial stretching of porcine thoracic aortas (PTAs). PTAs were sliced into approximately 50-\(\upmu \hbox {m}\)-thick sections, and the center of the sections was made especially thin using our previously developed technique. Alpha-elastin and cell nuclei were fluorescently labeled as indices of local elastin density and as a strain marker, respectively. Birefringence and second harmonic generation (SHG) light images were used to determine local collagen distributions. The specimens were then stretched biaxially with a laboratory-made tensile tester under a fluorescent microscope equipped with a birefringence imaging system. Local strains were calculated from the local displacement of the cell nuclei. The degree of alignment and density of local collagen fibers were measured from retardance and SHG images. The strain distributions, specifically the first and second principal, and maximum shear strains, fluorescent intensity of \(\upalpha \)-elastin, and degree of alignment of collagen fibers, showed insignificant differences between the crack initiation sites and other sites. The retardance and intensity of SHG light at the crack initiation sites were significantly lower than those at other sites for all (\(n = 6\)) specimens. Cracks tended to propagate along the local direction of the collagen fibers. These results indicate that the local density and direction of collagen fibers play an important role in aorta rupture.