Search

In the Japan Proton Accelerator Research Complex (J-PARC) linac, negative hydrogen beams are accelerated from 190 MeV to 400 MeV by twenty-one Annular-ring Coupled Structure (ACS) accelerating cavities. The input coupler of the ACS high-beta cavity, which is the 21st accelerating cavity (ACS21) in the order of beam acceleration, had a comparatively larger value of the Voltage Standing Wave Ratio (VSWR) than those of the other ACS cavities. To adjust the VSWR of the ACS21, we designed and fabricated a rectangular waveguide with a capacitive iris which conduces to a better matching between the cavity and the waveguide. In the 2018 summer maintenance period, we installed the newly fabricated waveguide to the ACS21 in the position between the input coupler and the RF window. Consequently, the VSWR of the ACS21 was successfully decreased to the target value which leads to the critical coupling under the nominal accelerating condition with 50-mA peak beam current., Proceedings of the 10th Int. Particle Accelerator Conf., IPAC2019, Melbourne, Australia

Annular-ring coupled structures (ACSs) will increase the beam energy of the Japan proton accelerator research complex (J-PARC) linac from 181 to 400 MeV to achieve a beam power of 1 MW for a materials and life science experimental facility. The mass production of the ACS cavities commenced in March 2009. Before the installation, all cavities require power testing. High-power testing is essential not only for confirming the cavity’s design performance but also for preventing delays in cavity conditioning schedule. However, the 2011 Tohoku earthquake damaged J-PARC facilities, including the ACS power-test area, and cavity conditioning was interrupted for two years. After the facility’s restoration, two ACS cavities (M01 and M11) were conditioned. They performed 15–20% above the designed accelerating field of 4.2 MV/m. As M01 was initially conditioned six years ago, the most recent conditioning time required for M01 was drastically reduced. From this result, we confirmed that long-term stored ACS cavities purged with nitrogen gas do not produce critical cavity performance issues. During high-power operation of M11, which is a unique cavity equipped with a capacitive iris in a waveguide, no significant increases in the temperature and the discharge rate around the capacitive iris were observed. Even considering beam loss due to residual gas scattering, the vacuum pressure was sufficiently low (4 × 10−6 Pa). More stable operation can be expected following a month-long conditioning process before the beam is commissioned. M11’s conditioning successfully demonstrated an auto-conditioning program, and we established the conditioning scheme using this auto-conditioning program for all ACS cavities in a limited time and with limited manpower.

The beam energy of the Japan Proton Accelerator Research Complex (J-PARC) linac has been increased from 181 to 400 MeV in January 2014. For the energy upgrade, the 25 Annular-ring Coupled Structure (ACS) cavities were installed in the beam transport at the downstream of the 191-MeV drift tube linac in 2013 maintenance period. To install and condition all the ACS cavities in a limited period of time, the upgrade of related systems such as a vacuum, magnets, RF, and cooling water has been started in 2010 and conducted in the annual maintenance period in every summer. This work includes the recovery efforts from the Tohoku earthquake in 2011. In this paper, the installation work for the energy upgrade and the related equipment for the ACS will be presented.

The accelerating cavities of the J-PARC linac, additionally comprising an annular-ring-coupled structure (ACS), went into operation in 2014. To further improve the vacuum pressure of the ACS, an additional nonevaporable getter (NEG) pump was designed so that it could be installed independent of the vacuum chamber of the ACS cavity. We confirmed that the NEG pump can be appropriately activated by using a small pumping station and that purging with noble gases reduces the saturation of the NEG surface. In the evacuation test of the prototype ACS cavity with the NEG pump, the partial pressure of H2 and the total pressure were reduced from 4.8×10−7 and 6.8×10−7 Pa to 2.5×10−7 and 4.5 × 10−7 Pa, respectively. The additional NEG pump will be installed in the ACS cavity in the fall of 2014, after which any decrease in pressure and NEG-pump lifetime will be confirmed by long-term-operation experiments.

Kazuo HASEGAWA*, Hidetomo OGURI, Takashi ITO, Etsuji CHISHIRO, Koichiro HIRANO, Takatoshi MORISHITA, Shinichi SHINOZAKI, Hiroyuki AO, Kiyonori OHKOSHI, Yasuhiro KONDO, Jun TAMURA, Saishun YAMAZAKI, Toshihiko HORI, Fumiaki SATO, Yasuo NEMOTO, Isao KOIZUMI, Nobuo OUCHI, Nobuhiro KIKUZAWA, Akira UENO, Akihiko MIURA, Shinpei FUKUTA, Akinobu YOSHII, Koichi SATO, Akira OZONE, Yuki SAWABE, Yusuke KAWANE, Hiroshi IKEDA, Yuichi ITO, Yuko KATO, Kazuo KIKUCHI, Fumio HIROKI, Toshio TAKAYASU, Tsutomu USAMI, Munetoshi YANAI, Kazuhiko TADOKORO, Kenji OHSAWA, Fujio NAITO, Yong LIU, Zhigao FANG, Takashi SUGIMURA, Kenta FUTATSUKAWA, Kiyoshi IKEGAMI, Masato KAWAMURA, Kesao NANMO, Yuji FUKUI, Tomoaki MIYAO, Tomofumi MARUTA and Akira TAKAGI

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.

The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

The synaptojanins represent a subfamily of inositol 5′-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

Synaptojanin (synaptojanin 1) is a recently identified inositol 5′-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an amino-terminal SacI homology region, a central inositol 5′-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predictedM r of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5′-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments. Our results demonstrate the presence of a family of synaptojanin-type inositol 5′-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.

The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I. The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH2-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radioiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides GT1b or GD1a. In synapses, the intravesicular NH2-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH2-terminal domain of synaptotagmin II forms binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

The protein receptor for Clostridium botulinum type B neurotoxin was purified 340-fold from rat synaptosomes by successive chromatography on DEAE-Sepharose, phenyl-Toyopearl, and heparin-Toyopearl columns. 125I-Labeled neurotoxin bound to lipid vesicles containing the protein receptor and ganglioside GT1b or GD1a. The reconstituted receptor showed the same affinities as the native receptor on synaptosomes. Chemical cross-linking of 125I-toxin to the receptor in the presence of gangliosides resulted in formation of a cross-linked product of 161 kDa under reducing conditions. Cross-linking was specific, as it was inhibited by the presence of excess unlabeled toxin. A monoclonal antibody against the purified 58-kDa receptor protein and a monoclonal antibody against the heavy chain (103 kDa) of the neurotoxin reacted with the cross-linked product of 161 kDa in immunoblotting experiments. We determined partial amino acid sequences of the 58-kDa protein, which were identical to synaptotagmin, a synaptic vesicle membrane protein. In addition, the monoclonal antibody against the 58-kDa receptor protein recognized recombinant rat synaptotagmin. These results suggest that synaptotagmin in association with ganglioside GT1b or GD1a may be a natural receptor for C. botulinum type B neurotoxin at the nerve terminals.

Using an antibody raised against an odor receptor protein, we investigated immunohistochemically the spatial distribution in the embryonic and adult rat olfactory epithelium of the olfactory receptor neurons that express the odor receptor protein. In adults, the immunoreactive olfactory receptor neurons were intermingled with immuno-negative receptor neurons, but were mostly restricted within a circumferential zone located in the lateral part of the epithelium. The immunoreactive olfactory receptor neurons were observed as early as embryonic day 14, with a strong tendency to localize in the lateral part of the epithelium. These results indicate that both selection of the odor receptor protein by individual olfactory receptor neurons and zonal segregation of the odor receptor protein expression occur early in embryonic development of the olfactory system.

R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons. Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb. We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons. Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase). Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen. R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin. R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively. Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system. Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction. Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

Botulinum C3 exoenzyme was used to specifically ADP-ribosylate and inactivate rho p21, and the effects of rho p21 inactivation on lysophosphatidic acid (LPA)-induced tyrosine phosphorylation were examined in cultured Swiss 3T3 cells. LPA induced a rapid increase in the tyrosine phosphorylation of a number of proteins. Pretreatment of the cells with the C3 exoenzyme caused ADP-ribosylation of rho p21 in the cells and selectively attenuated the phosphorylation of several proteins, including p43 mitogen-activated protein kinase, p125 focal adhesion kinase, and two proteins of 72 and 88 kDa. C3 exoenzyme pretreatment did not block the initial phosphorylation and activation of mitogen-activated protein kinase but suppressed its subsequent rise. In contrast, the enzyme treatment inhibited the induction of phosphorylation of the 72- and 88-kDa proteins and suppressed the basal and LPA-induced tyrosine phosphorylation of p125 focal adhesion kinase. In addition, immunoprecipitation of cell lysates with an antibody directed against the 85-kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase) co-precipitated a tyrosine-phosphorylated band of 180 kDa. C3 exoenzyme pretreatment suppressed both the phosphorylation of this band and PI 3-kinase activation associated with LPA stimulation. These findings suggest that rho p21 works as a link between the LPA receptor signal and the subsequent tyrosine phosphorylation and PI 3-kinase activation in these cells.

海面は廃棄物処分場用地の確保に苦しんでいる自治体等から熱い視線が向けられている。しかし, 海面は波浪等自然条件の影響が大きく処分場の適地は極めて限られている。また, 浸出水の処理を埋立終了後も長期にわたって行わなければならないなど解決しなければならない課題も多い。新たな処分場を設置する際には, 埋立対象物を環境に及ぼす影響が少なく, 土地としての価値が高くなるよう無害化処理したものに限定すること, 埋立中から海洋生物の成育環境への配慮や港湾施設及びマリンスポーツ施設等と共存することのできる処分場とすることが必要である。

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P 2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

Publisher Summary A widely accepted model describes the synaptic vesicle cycle as a modification of the receptor-mediated recycling pathway present in all cells. This pathway, by which cell surface receptors, like transferrin or low-density lipoprotein receptors, are internalized and recycled back to the surface, involves two distinct vesicular transport steps: clathrin-mediated budding from the plasma membrane and fusion with early endosomes and budding from endosomes of vesicles destined to the plasma membrane. The membrane composition of synaptic vesicles and of clathrin-coated vesicles purified from synaptosomes is very similar. The clathrin adaptor complex AP2, a component of the plasma membrane-derived clathrin coat, binds to synaptotagmin. The paralytic shibire Drosophilu mutants , whose nerve terminals are depleted of synaptic vesicles at the restrictive temperature due to selective impairment of synaptic vesicle endocytosis, harbor mutations in dynamin, a guanosine triphosphatase (GTPase) implicated in clathrin-mediated endocytosis. Fission of clathrin-coated pits to form free vesicles requires dynamin. Dynamin was originally described as a microtubule-binding protein, but its function in synaptic vesicle recycling was revealed when the similarity to the protein encoded by the Drosophilu gene shibire was discovered. Until recently, most studies on synaptic vesicle recycling have focused on the role of proteins. There is increasing evidence, however, for a role of lipids and of phosphoinositides in particular, in synaptic vesicle endocytosis.

SYNAPTOJANIN is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling1,2. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe3–7. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sacl (Rsdl), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton8–10. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphi-physin, a presynaptic protein with a putative function in endo-cytosis11–14. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.

Several proteins have been postulated as possible targets of immune attack in Lambert-Eaton myasthenic syndrome (LEMS). Heterogeneity of autoantibodies in sera from 20 LEMS patients was studied by comparing their reactivity to synaptotagmin, a synaptic vesicle protein, and voltage-gated calcium channels (VGCCs). Six patients' sera (1 with small cell lung carcinoma (SCLC)) contained antibodies specifically recognizing the recombinant synaptotagmin on immunoblots. Thirteen (11 with SCLC) and 16 (11 with SCLC and 1 with poorly differentiated cell carcinoma in the lung) patients' sera immunoprecipitated ω-conotoxin GVIA-labeled N-type and (ω-conotoxin MVIIC-labeled Q-type VGCCs, respectively. Three of 6 synaptotagmin-positive sera had cross-reactivity with N and/or Q subtypes of VGCC; the remaining 3 showed no cross-reactivity with VGCCs. Results indicate that LEMS sera are heterogeneous in the spectrum of containing antibodies, and suggest that this heterogeneity reflects the immune response to various synaptic proteins including not only multiple VGCCs but also synaptosecretory complex proteins.

Clostridium botulinum C3 exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp18 and Glu173 of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu173 caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18 remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18 substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173-substituted enzyme. These results indicate that the Glu173 residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.

Telencephalin (TLN) is a 130 kd glycoprotein expressed exclusively in neurons of the telencephalon, the most rostral brain segment. In the neurons, TLN is localized to soma-dendritic membrane but not to axonal membrane. In this study, we have cloned cDNA encoding rabbit and mouse TLN. The cDNA-derived primary structure of TLN predicts an integral membrane protein with nine tandem immunoglobulin-like domains in an extra-cellular region, a transmembrane domain, and a short cytoplasmic tail. The distal eight immunoglobulin-like domains of TLN show highest homology with the immunoglobulin-like domains of intracellular adhesion molecules (ICAMs) 1, 2, and 3/R. The structural similarity of TLN with ICAMs provides a new and strong link between immunoglobulin superfamily molecules in the nervous and immune systems. TLN is an example of a dendrite-associated cell adhesion molecule involved in the brain's segmental organization, cell-cell interactions during dendritic development, and maintenance of functional neuronal networks.

6R-L-Erythro-5,6,7,8-Tetrahydrobiopterin (R-THBP) is a common cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases, the latter two of which are the rate-limiting enzymes of catecholamines and serotonin biosyntheses, respectively. In an attempt to answer the puzzle from the clinical evaluation that R-THBP and L-DOPA or 5-HTP possess the additive effect on the treatment of atypical phenylketonuria, Parkinsonian disease, and infantile autism, we recently found a novel role of R-THBP in the central nervous system; R-THBP functions as a release-promotor of dopamine, serotonin, and norepinephrine from the striatal and cortical nerve terminals1,2. Amino acid neurotransmitters in the extracellular space also increased by R-THBP treatment, but in this case, the destruction of catecholaminergic components by 6-hydroxydopamine pretreatment resulted in a complete blockade of amino acid release evoked by R-THBP2. Namely, the effect of R-THBP on amino acid neurotransmitters is via monoaminergic stimulation. R-THBP also evokes acetylcholine release possibly through serotonergic stimulation3. On the contrary, when we reduce R-THBP content in the tissues including the brain by use of a potent inhibitor of R-THBP biosynthesis, the tissue content of dopamine and its metabolites very slightly decreased at around 10%, but such a treatment lowering R-THBP level resulted in a severe decrease of dopamine release; the dopamine output from the striatum was reduced to less than 50% of the control level. This result and other experimental data indicate that the endogenous level of R-THBP can regulate dopamine release. The molecular mechanisms underlying the R-THBP-induced neurotransmitter amine release have been studied in terms of second messenger system and modification of autoreceptor system for presynaptic dopamine terminals4. In the course of the study, the high-affinity binding protein specific for [3H]R-THBP has been found in the crude membrane fraction of rat brain. However, the binding activity was also found in the cytosol fraction. Here, we describe several lines of evidence showing that the binding protein in the cytosol is almost entirely due to nitric oxide synthase.

Summary A 1.5 kilobase cDNA coding for the complete amino acid sequence of Gb, the substrate for ADP-ribosyltransferase in C1 and D botulinum toxins from bovine adrenal gland, has been isolated from a cDNA library of bovine adrenal gland. This cDNA encodes a polypeptide of 21,770 Da consisting of 193 amino acid residues, and the deduced amino acid sequence contains all the partial amino acid sequences reported previously ( Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. , 263 , 17255–17257). Sequence comparison revealed that Gb is identical with the product of human rho clone 12 ( rho A). The present results also confirmed our suggestion that the ADP-ribosylation occurs at Asn 41 in the putative effector domain of the rho gene product.

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing RhoGAP1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.

Poly(ADP-ribosyl)ation constitutes a novel type of posttranslational covalent modification of nuclear proteins. The synthesis of poly(ADP-ribose) is catalyzed by poly(ADP-ribose) synthetase (1). Recently, poly(ADP-ribose) groups on proteins were found to turn over rapidly in vivo (2, 3). The degradation of poly(ADP-ribose) is carried out by two different types of enzymes. ADP-ribosyl protein lyase cleaves the bond between mono(ADP-ribose) and protein (4). Another enzyme is poly(ADP-ribose) glycohydrolase, which is known to split the ribose-ribose glycosidic bond (5, 6). The glycohydrolase is the major enzyme responsible for polymer degradation in vivo (7). Therefore, the knowledge of its precise mode of action on poly(ADP-ribose) is important for elucidating not only the metabolism of poly(ADP-ribose) but also the biological functions of poly(ADP-ribosyl)ation. In the present study, we purified this glycohydrolase, and investigated its mode of action precisely.

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.