Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron.

Synaptotagmin I or II (Syt I/II) is involved in Ca2+-regulated exocytosis of secretory vesicles, probably serving as a Ca2+-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca2+-regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune immunoglobulin G had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.

Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth.

Synaptotagmin I (or II), a possible Ca(2+)-sensor of synaptic vesicles, has two functionally distinct C2 domains: the C2A domain binds Ca2+ and the C2B domain binds inositol high polyphosphates (IP4, IP5, and IP6). Ca(2+)-regulated exocytosis of secretory vesicles is proposed to be activated by Ca2+ binding to the C2A domain and inhibited by inositol polyphosphate binding to the C2B domain. Synaptotagmins now constitute a large family and are thought to be involved in both regulated and constitutive vesicular trafficking. They are classified from their distribution as neuronal (synaptotagmin I-V, X, and XI) and the ubiquitous type (synaptotagmin VI-IX). Among them, synaptotagmins III, V, VI and X are deficient in IP4 binding activity due to the amino acid substitutions in the C-terminal region of the C2B domain, suggesting that these isoforms can work for vesicular trafficking even in the presence of inositol high polyphosphates. Synaptotagmin I is also known to be present in neuronal growth cone vesicles. Antibody against the C2A domain (anti-C2A) that inhibits Ca(2+)-regulated exocytosis also blocked neurite outgrowth of the chick dorsal root ganglion (DRG) neuron, suggesting that Ca(2+)-dependent synaptotagmin activation is also crucial for neurite outgrowth.

The alteration of nasal resistance before and after local exposure to heated aerosol in perennial allergic rhinitis.

To determine the patho-physiological effects of heated vapour to the normal or allergic nasal mucosa, we measured the nasal resistance before and after a 10 min. exposure of hyperthermal (43.0 degrees C) aerosol to the nasal mucosa in normal subjects and perennial allergic rhinitis patients. In the allergic patients the mean nasal resistances after hyperthermal stimulation were significantly higher than those resistances without stimulation, both in expiration or inspiration. No significant differences of nasal resistances in normal individuals during the whole schedule with and without heated aerosol stimulation were found on expiration or inspiration. The local heated aerosol exposure increases the nasal resistance in nasal allergic patients while in normal subjects no changes were found, and the reaction may have arisen from a non-specific hypersensitivity of the susceptible allergic nasal mucosa.

The distribution of eosinophil cationic protein positive eosinophils in the nasal mucosa of the nasal allergy patients.

The relationship between the distribution of eosinophils and epithelial damage of the nasal mucosa in nasal allergy was investigated by means of hematoxylin and eosin staining and a technique of immunohistochemistry using the anti-human EG2 mouse monoclonal antibody that reacts with the secreted form of eosinophil cationic protein (ECP). Nasal mucosa tissue of 26 adult nasal allergy patients and of 24 adult non-allergic rhinitis patients was removed surgically. Eosinophilia in the nasal mucosa of the allergy group was greater than that in the non-allergy group. A great number of ECP positive eosinophils accumulating in the nasal mucosa of the allergy group were mostly degranulated at the superficial layer of the lamina propria. Desquamation of the epithelium was observed mainly in the area of eosinophilia with degranulation of the secreted form of ECP in the nasal allergy group.

Seasonal variations of nasal resistance in allergic rhinitis and environmental pollen counts.

We made measurements of nasal airway resistance in a patient with Japanese cedar pollinosis and also measured the environmental pollen counts under different exposure conditions. In the season of high Japanese cedar pollen counts in Japan values of mean nasal airway resistance were significantly increased, remained elevated for 2 months after the season, and eventually decreased. In the season of low pollen counts, the mean values of nasal airway resistance measurements were increased during the season but decreased more rapidly after the season. In a year with no exposure to the environmental allergen because the subject lived out of Japan, the mean nasal airway resistances were relatively increased closely in phase with the Japanese cedar pollen season.

Seasonal variations of nasal resistance in allergic rhinitis and environmental pollen counts. III: Efficacy of antiallergic agents.

We previously reported that, in a patient with Japanese cedar pollinosis, preseasonal increase in total nasal airway resistance (NAR) possibly caused from circannual endogenous rhythm was suppressed by preseasonal systemic administration of antiallergic agent. In this study, in order to determine the influence of preseasonal use of a topical antiallergic agent and seasonal administration of a peroral antiallergic agent, we made measurement of nasal airway resistance and pollen counts under the previous respective conditions in separated years. Preseasonal high mean values of total NAR for a couple of weeks was not suppressed by the preseasonal topical administration of ketotifen nasal spray in 1992 which was a low pollen season (974/cm2). However, the patient did not complained of severe episodes of hay fever at all throughout the season. Preseasonal increases in mean values of total NAR were observed in 1993 which was a high Japanese cedar pollen season (4,875/cm2) with NAR the same as untreated seasons. This may have been because the patient was not treated with preseasonal use of peroral azelastine. On extremely high Japanese cedar pollen count days further increased NAR occurred in the patient. The patient experienced relatively severe hay fever episodes, but used no other anti-histamine nor topical steroid during the season.

A unique spacer domain of synaptotagmin IV is essential for Golgi localization.

Synaptotagmin (Syt) family members consist of six separate domains: a short amino terminus, a single transmembrane domain, a spacer domain, a C2A domain, a C2B domain and a short carboxyl (C) terminus. Despite sharing the same domain structures, several synaptotagmin isoforms show distinct subcellular localization. Syt IV is mainly localized at the Golgi, while Syt I, a possible Ca(2+)-sensor for secretory vesicles, is localized at dense-core vesicles and synaptic-like microvesicles in PC12 cells. In this study, we sought to identify the region responsible for the Golgi localization of Syt IV by immunocytochemical and biochemical analyses as a means of defining the distinct subcellular localization of the synaptotagmin family. We found that the unique C-terminus of the spacer domain (amino acid residues 73-144) between the transmembrane domain and the C2A domain is essential for the Golgi localization of Syt IV. In addition, the short C-terminus is probably involved in proper folding of the protein, especially the C2B domain. Without the C-terminus, Syt IVdeltaC proteins are not targeted to the Golgi and seem to colocalize with an endoplasmic reticulum (ER) marker (i.e. induce crystalloid ER-like structures). On the basis of these results, we propose that the divergent spacer domain among synaptotagmin isoforms may contain certain signals that determine the final destination of each isoform.

Inositol 1,3,4,5-tetrakisphosphate binding activities of neuronal and non-neuronal synaptotagmins. Identification of conserved amino acid substitutions that abolish inositol 1,3,4,5-tetrakisphosphate binding to synaptotagmins III, V, and X.

Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.

XynX, a possible exo-xylanase of Aeromonas caviae ME-1 that produces exclusively xylobiose and xylotetraose from xylan.

A gene, xynX, encoding a novel xylanase, was cloned from Aeromonas caviae ME-1. This gene encoded an enzyme that was constituted of 334 amino acid residues (38,580 Da) and was similar in sequence to Family 10 (Family F) beta-1,4 endo-xylanases. XynX produced only xylobiose and xylotetraose from birch wood xylan, and xylotriose, xylopentaose, and higher oligosaccharides were not detected in the TLC analysis. We designated it as X2/X4-forming xylanase. This enzyme does not have transglycosylation activity. These data suggested that this enzyme is a possible exo-xylanase. According to homology modeling, the enzyme has a ring-shaped (alpha/beta)8 barrel (TIM barrel) structure, typical of Family 10 endo-xylanases, with the extraordinary feature of a longer bottom-loop structure.

Long-chain betulaprenol-type polyprenols from the leaves of Ginkgo biloba.

A long-chain betulaprenol-type polyprenol mixture was isolated from the leaves of Ginkgo biloba mainly as acetate. The structure was determined by mass spectroscopy, 1H-n.m.r. spectroscopy and 13C-n.m.r. spectroscopy. The mixture contained polyprenols-14-22, predominantly polyprenols-17, -18 and -19, and consisted of the dimethylallyl terminal unit (omega-terminal), two trans-isoprene residues, a sequence of 11-19 cis-isoprene residues and a terminal hydroxylated isoprene unit (alpha-terminal) aligned in that order. The concentration of these polyprenols in leaves increased from 0.04 to 2.0% of dry wt. with maturing of the leaves, though the content of total lipids was constant. The distribution of chain length in these polyprenols showed little variation throughout the whole life of the leaves.

Cloning, expression, and characterization of a family 52 beta-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1.

A lambda phage genomic library of Aeromonas caviae ME-1, a multiple-xylanase-producing bacterium, was screened for xylan degradation activities. We isolated one clone, B65, which had weak xylanase activity, by the DNS method, but gave no visible bands on zymogram assay using SDS-xylan-PAGE. Based on TLC analyses of enzymatic products and some glycosidase assays using p-nitrophenyl substrates, we established that pB65 encodes a beta-xylosidase gene. In the nucleotide sequence analysis, we found a 2190-bp open reading frame (ORF) named xysB. XysB protein is similar to some beta-xylosidases, which are categorized in the glycosyl hydrolase family 52. Another ORF (xyg), that showed similarity to the family 67 alpha-glucuronidase, was also found downstream of the xysB gene. The xysB ORF and its promoter region were cloned into the pT7-Blue vector and the transformant cells had beta-xylosidase activity. The relative molecular mass were estimated to be 75 kDa by SDS-PAGE and 159 kDa by gel filtration. These data showed that XysB has a dimeric structure of 80,697 Da subunits. This enzyme showed optimal activity at 50 degrees C and pH 6.0. It was stable below 40 degrees C and pH 5-8. The Km and Vmax were calculated to be 0.34 mM and 33 nmol x min(-1) x microg(-1), respectively. This enzyme also showed transglycosylation activity against X3 and produced X4 and X5.

Synaptotagmin IV is present at the Golgi and distal parts of neurites.

Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor-differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform-specific antibody (anti-SytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A-dependent gene up-regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity.

SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms.

Synaptotagmins (Syts) are a large family of membrane proteins consisted of at least 12 isoforms. They are categorized in neuron-specific isoforms (I-V, X, and XI) and ubiquitous isoforms (VI-IX) based on their expression patterns. Syt-I, a neuron-specific and abundant isoform, has been well characterized and postulated to be the exocytotic Ca(2+) sensor. However, the functions of other isoforms remain obscure. Here, we report that ubiquitous isoforms of synaptotagmins, Syt-VII, Syt-VIII, and Syt-IX, interacted with a cytoplasmic RNA-binding protein, SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein), through their C2B domains. SYNCRIP was originally found in the Syt-II C2AB domain bound fraction from the mouse brain lysate. cDNA cloning of SYNCRIP cDNA revealed that the protein was highly homologous to heterogeneous nuclear ribonucleoprotein R (hnRNP R) recently identified. SYNCRIP protein was ubiquitously and constantly expressed in various tissues of mice parallel to hnRNP R. SYNCRIP indeed bound RNA with preference to poly(A) RNA; however, in contrast to the nuclear localization of hnRNP R, SYNCRIP was distributed predominantly in the cytoplasm as judged by both biochemical fractionation and immunohistochemical studies. In vitro binding experiments showed the potential interaction of SYNCRIP with C2B domains of Syts except for those of Syt-V, -VI, and -X. Furthermore, the interaction between SYNCRIP and Syt-VII, -VIII, or -IX was revealed by co-immunoprecipitation experiments using COS cells transiently expressing each Syt isoform. These findings suggested that SYNCRIP was a target of ubiquitous type of Syts and implied the involvement of ubiquitous Syts in the regulation of dynamics of the cytoplasmic mRNA.