Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.     

The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).     

Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.     

Ownership-dependent mating tactics of minor males of the beetle <Emphasis Type="Italic">Librodor japonicus</Emphasis> (Nitidulidae) with intra-sexual dimorphism of mandibles

Intra-sexual dimorphism is found in the weapons of many male beetles. Different behavioral tactics to access females between major and minor males, which adopt fighting and alternative tactics, respectively, are thought to maintain the male dimorphism. In these species major males have enlarged weapons that they use in fights with rival males. Minor males also have small weapons in some of these species, and it is unclear why these males possess weapons. We examined the hypothesis that minor males might adopt a fighting tactic when their status was relatively high in comparison with that of other males (e.g., ownership of a territory). We observed the behavioral tactics of major and minor males of the beetle Librodor japonicus, whose males have a dimorphism of their mandibles. Major males fought for resources, whereas minor males adopted two status-dependent tactics, fighting and sneaking, to access females, depending on their ownership of a sap site. We suggest that ownership status-dependent mating tactics in minor males may maintain the intra-sexual dimorphism in this beetle.     

Crystal structure of Ufc1, the Ufm1-conjugating enzyme

Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.     

CP5484, a novel quaternary carbapenem with potent anti-MRSA activity and reduced toxicity

A new series of 1beta-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and the activities of these compounds against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. To study the effect of basic moieties on anti-MRSA activity, we introduced an amino, or imino, or amidino group at the 6-position of imidazo[5,1-b]thiazole in place of the carbamoylmethyl moiety of CP5068. Anti-MRSA activities of almost all basic group-substituted carbapenems were improved, though some of the compounds showed stronger acute toxicity in mice than IPM. In order to decrease the toxicity without decreasing the activity, we introduced various additional functionalities around the basic moiety. Finally, we obtained CP5484, which has excellent anti-MRSA activity and low acute toxicity.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

New evidence of reproductive organs of <Emphasis Type="Italic">Glossopteris</Emphasis> based on permineralized fossils from Queensland,Australia. I. Ovulate organ <Emphasis Type="Italic">Homevaleia</Emphasis> gen. nov.

This study describes Homevaleia gouldii H. Nishida, Pigg, Kudo et Rigby gen. et sp. nov., an ovule-bearing glossopterid organ, based on a combination of recently collected permineralized specimens from the Late Permian Homevale Station locality in the Bowen Basin of Queensland, Australia, and on previously studied material from the 1977 Gould and Delevoryas study. Homevaleia, which resembles the compression–impression genus Dictyopteridium, is an inrolled megasporophyll with a distinct keel that bears numerous (over 70) stalked ovules on its adaxial surface. Ovules are small, oval, with an elaborate mesh-like structure that is developed from the outermost integumentary layers. Specimens interpreted as representing different developmental stages show there is an apparent interrelationship between megagametophyte development and the opening of the surrounding fertile structure for pollination. Together, new information provided by this material enables better understanding of glossopterid reproductive structure and its function in one distinctive form.