Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.     

A water quality analysis system to evaluate the impact of agricultural activities on N outflow in river basins in Japan

We have developed a personal-computer-based water quality analysis system for river basins. The system estimates potential N outflow by model and calculates actual N outflow from monitoring data. For the former it uses the potential load factor method to estimate annual nitrogen load from various sources and runoff potential from each area of land in a basin. For the latter it analyzes water quality monitoring data in relation to meteorological data. We used the system to analyze N outflow in basins around Lake Kasumigaura and the Yahagi River in central Honshu, Japan. The land around Lake Kasumigaura is rather flat, and about 25% is periodically flooded for rice and lotus cultivation. The land around the Yahagi River is mountainous, and much less land is flooded. In the Yahagi River basin the actual N outflow agreed closely with the potential. However, the actual N outflow in the basin around Lake Kasumigaura was much less than the potential, suggesting that a large part of the N load is denitrified in flooded soils. This further indicates that a sequence of different land uses including flooded rice fields is an important factor determining N outflow in basins in Japan. On the basis of the above analyses, we incorporated a denitrification model into the system that enables us to estimate N balance in a designated basin;this system may be helpful in the formulation of scenarios of land use andsoil management for improving water quality.     

A novel method for construction of gene fragment library to searching epitopes

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.     

Complete Genome Sequence of the Wild-Type Commensal Escherichia coli Strain SE15, Belonging to Phylogenetic Group B2

Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism.The complete genome sequence of Escherichia coli SE15 was determined using a combination of 2-kb and 40-kb Sanger libraries and 454 pyrosequencing. We generated 57,600 sequences (ABI 3730xl sequencers) and three sequencing runs (GS20 sequencers). The 454 pyrosequencing reads were first assembled using the Newbler assembler software (4). A hybrid assembly of 454 and Sanger reads was performed using the Phred-Phrap-Consed program (1). Remaining gaps between contigs were closed by direct sequencing of clones. Prediction and annotation of protein-coding genes were performed as described previously (6).The genome of E. coli SE15 consists of a circular 4,717,338-bp chromosome containing 4,338 predicted protein-coding genes and a 122-kb plasmid (pSE15) encoding 150 protein-coding genes. From the multilocus sequence typing analysis based on the nucleotide sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), SE15 was found to belong to E. coli reference collection group B2. In the chromosome, two prophage regions and seven integrative elements are found. Of the predicted protein-coding genes, we could assign 2,883 (64%) to known functions, 1,528 (34%) as conserved hypothetical genes and 77 (2%) as novel hypothetical genes. Of the predicted protein-coding genes on the chromosome, 3,735 (86%) are common to three uropathogenic E. coli (UPEC) genomes (CFT073, UTI89, and 536) and 263 (6%) are not identified in any of the three UPEC genomes. The 263 genes include 7 genes for the phosphoenolpyruvate:sugar phosphotransferase system involved in the uptake of carbohydrates, reflecting the adaptation of SE15 to a commensal lifestyle in the intestinal tract. pSE15 shares 121 genes (81%) with a 114-kb plasmid (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CP000244,term_id:91075696,term_text:CP000244}}CP000244) of UPEC UTI89, indicating that both plasmids are derived from the same origin.The chromosome contains six large segments (LSs; >30 kb) designated LSs I to VI, three of which overlap one prophage region and two integrative elements. Each of the six LSs is located at the same locus as at least one of the pathogenicity islands (PAIs) or other insertion regions in the three UPEC genomes. LS II (ECSF_1824 to ECSF_1835) and three PAIs (PAI IV_UTI89, PAI IV₅₃₆, and HPI_CFT073) are located at the same loci in each chromosome and share the ybt operon encoding the yersiniabactin iron acquisition system, indicating that the ancestral E. coli of group B2 strains may have acquired the ybt genes. LS III (ECSF_1852 to ECSF_1897), PAI VI_UTI89, PAI VI₅₃₆, and PAI_CFT073-asnW are located at the same loci in each chromosome. The three PAIs contain the pks island encoding multiple nonribosomal peptide synthases and polyketide synthases, whereas LS III in SE15 completely lacks the pks island. The commensal E. coli strain ED1a also lacks the pks island (8), but the commensal E. coli strain Nissle 1917 has the pks island (5). These data suggest that the presence of the pks island may not be common among intestinal commensal strains in group B2. LS V (ECSF_2770 to ECSF_2794) is almost identical to PAI V_UTI89, which contains the genes cluster for a type II secretion system (gsp), group II capsule synthesis (kps), and polysialic acid synthesis (neu). The neu operon between the kpsFEDUCS and kpsMT genes in PAI V_UTI89 is responsible for K1 capsule biosynthesis, and this region between the kpsFEDUCS and kpsMT genes is highly variable in E. coli (9). The corresponding region (ECSF_2777 to ECSF_2781) in LS V encodes genes different from those in the neu operon in PAI V_UTI89; differs from the corresponding regions of the CFT073 (K2 serotype), 536 (K15 serotype), and APEC O1 (K1 serotype) strains; and shows no homology with any sequence in public databases.SE15 lacks many virulence-related genes, whereas UPEC encodes virulence-related factors, including fimbrial adhesins, toxins, capsule, and serum resistance and iron uptake systems. The three UPEC strains have the genes encoding P fimbriae (pap), S fimbriae (sfa/foc), Auf fimbriae (auf), and type 1 fimbriae (fim), whereas SE15 contains only the fim genes and lacked the pap, sfa/foc, and auf genes. Amino acid replacements in FimH located at the tip of type 1 fimbriae produce a shift from a commensal-associated trimannose binding phenotype to a urinary tract infection-associated monomannose binding phenotype (7). The other sequenced B2 strains (three UPEC strains, APEC O1, LF82, and ED1a) have Ser-70 and Asn-78 residues in FimH, whereas SE15 has Asn-70 and Ser-78 residues that are conserved in intestinal E. coli strains. Of the seven chaperon-usher fimbrial operons in SE15, six (fim, yad, yde, yeh, yfc, and yqi) are conserved in the three UPEC genomes. The one remaining fimbrial operon (ECSF_0163 to ECSF_0166) is specific to SE15. The GC content (42%) of this 5-kb fimbrial region is lower than the average GC content (51%) of the chromosome. UPEC strains contain a greater number of iron acquisition systems than do commensal strains, which may be a consequence of their adaptation to the iron-limiting urinary tract environment (3). SE15 also contains iron uptake system genes encoding siderophore enterobactin, siderophore yersiniabactin, iron transporter (sit), and heme (chu) systems but lacks genes for siderophore salmochelin, siderophore aerobactin, and novel siderophore (ireA), which are encoded by PAIs of UPEC strains. Furthermore, SE15 lacks genes encoding alpha-hemolysin and cytotoxic necrotizing factor, which are known toxins encoded by PAIs of UPEC strains.It has been pointed out that extraintestinal pathogenic E. coli (ExPEC) virulence factors identified in commensal strains of group B2 may facilitate colonization of the human gut and thus act as fitness factors for commensal E. coli stains (2). SE15 contains fewer known ExPEC virulence-associated genes than other known commensal strains (ED1a and Nissle 1917) in group B2, suggesting that ExPEC virulence-related genes in the SE15 genome may be necessary for this commensal microorganism to colonize the human gut.     

Photooxidative cleavage of zinc 20-substituted chlorophyll derivatives: conformationally P-helix-favored formation of regioselectively 19-20 opened linear tetrapyrroles

Photoreaction of zinc methyl 20-substituted meso(pyro)pheophorbide-a prepared by modifying naturally occurring chlorophyll-a in the presence of oxygen molecules gave its C19-C20 oxidative cleavage (1-carbonyl-19-oxo-bilatrienes) as the major products and the regioisomeric C1-C20 cleavage (19-carbonyl-1-oxo-bilatrienes) as the minor products. The resulting zinc complexes of linear tetrapyrroles took a helical conformation and the P-conformers were preferential over the M-stereoisomers due to the presence of their 17S,18S-chiral centers. The helical conformers (diastereomers) of the corresponding nickel complexes were separated by reverse-phase or chiral HPLC and their conformational changes were observed in solution.     

STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM, BOMBYX MORI : I. Growth of Posterior Silk Gland Cells and Biosynthesis of Fibroin During the Fifth Larval Instar

Growth of the posterior silk gland and biosynthesis of fibroin during the fifth larval instar of the silkworm, Bombyx mori, have been studied. In accordance with the exponential increase in the wet weight of the gland, the amounts of DNA, RNA, protein, and lipids per animal increased rapidly in the early stage of the fifth instar (0–96 hr). Biosynthesis of fibroin, on the contrary, mainly proceeds in the later stage of the fifth instar (120–192 hr). Electron microscopical observations have shown that, in the very early stage (0–12 hr), a number of free ribosomes exist in the cytoplasm. Rough endoplasmic reticulum (ER) with closely spaced cisternae was also observed. Then rough ER starts to proliferate rapidly, and at the same time lamellar ER is rapidly or gradually transformed into vesicular or tubular forms. In the later stage of the fifth instar (120–192 hr), the cytoplasm is mostly filled with tubular or vesicular ER. Golgi vacuoles, free vacuoles (fibroin globules), and mitochondria are also observed. It is concluded that in the early stage of the fifth instar the cellular structures necessary for the biosynthesis of fibroin are rapidly formed, while in the later stage the biosynthesis of fibroin proceeds at a maximum rate and utilizes these structures.     

Exocytosis mechanism as a new targeting site for mechanisms of action of antiepileptic drugs

Carbamazepine (CBZ) and zonisamide (ZNS) are effective antiepileptic drugs (AEDs) for the treatment of epilepsy and mood disorder. One of the mechanisms of action of CBZ and ZNS is inactivation of voltage-gated Na+ channel (VGSC). However, the major mechanism(s) of action of these AEDs is not clear yet. We have been exploring novel targeting mechanisms for the antiepileptic actions of CBZ and ZNS during the past ten years. In this report, we describe our hypothesis regarding the new targeting mechanisms for the antiepileptic action of AEDs. We determined an interaction between these AEDs and inhibitors of both voltage-sensitive Ca2+ channels (VSCCs) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) on neurotransmitter exocytosis using microdialysis. Perfusion with therapeutic concentrations of CBZ and ZNS increased basal neurotransmitter release. This stimulatory action was predominantly inhibited by inhibitors of N-type VSCC and syntaxin. CBZ and ZNS increased Ca2+-evoked release, an action selectively inhibited by inhibitors of N-type VSCC and syntaxin. CBZ and ZNS reduced K+-evoked release, an action predominantly inhibited by inhibitors of P-type VSCCs and synaptobrevin. These actions of CBZ and ZNS on neurotransmitter exocytosis could be observed under the condition of inhibition of VGSC using perfusion with tetrodotoxin. Our findings enhance our understanding of the mechanisms of action of CBZ and ZNS as AEDs, which possibly reduce P-type VSCCs/synaptobrevin-related exocytosis mechanisms during the depolarization stage, and simultaneously enhance N-type VSCCs/syntaxin-related exocytosis mechanisms at the resting stage.     

Identification of enterohemorrhagic Escherichia coli O157-Specific DNA sequence obtained from amplified fragment length polymorphism analysis

An approximately 1.1 kbp fragment that was commonly observed only in the enterohemorrhagic Escherichia coli (EHEC) O157 strains in an analysis of amplified fragment length polymorphism was found to be a partial gene sequence encoding the locus of toxB and a useful molecular marker for the identification of EHEC O157.     

Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%–80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these “osteocyte-less” mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.