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.     

Localization of TGF-beta signaling intermediates Smad2, 3, 4, and 7 in developing and mature human and mouse kidney.

Smad proteins are signaling intermediates of the TGF-beta superfamily and are involved in a range of biological activities including development and immune responses. We studied the expression of TGF-beta-receptor activated Smads (Smad2 and Smad3), the common partner Smad (Smad4), an inhibitory Smad (Smad7), and the activated (phosphorylated) Smad2 (pSmad2) in developing and adult kidneys of humans and mice. These studies demonstrate associated expression of these Smads in multiple renal cell types in all developmental stages and in mature non-diseased kidneys. Smad expression is in general most widespread at the earliest stages of nephron development and diminishes as components of the nephrons become more differentiated. Paucity of Smad expression in mesangial cells in contrast to widespread expression of these Smads in glomerular visceral epithelial cells in both developing and mature kidneys was remarkable. Divergent and less extensive expression of Smad4, compared with other Smad proteins, was also demonstrated in tubules of human kidneys. Based on the observed expression patterns, these findings demonstrate, for the first time, expression of the TGF-beta-receptor-activated Smad2 and Smad3, the common mediator Smad4, and the inhibitory Smad7 in the developing human fetal kidney, extending observations previously made in rodent systems to humans.     

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.     

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.     

The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

Mesenchymal stem cell isolation and characterization from human spinal ligaments

Mesenchymal stem cells (MSCs) have a fibroblast-like morphology, multilineage potential, long-term viability and capacity for self-renewal. While several articles describe isolating MSCs from various human tissues, there are no reports of isolating MSCs from human spinal ligaments, and their localization in situ. If MSCs are found in human spinal ligaments, they could be used to investigate hypertrophy or ossification of spinal ligaments. To isolate and characterize MSCs from human spinal ligaments, spinal ligaments were harvested aseptically from eight patients during surgery for lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament. After collagenase digestion, nucleated cells were seeded at an appropriate density to avoid colony-to-colony contact. Cells were cultured in osteogenic, adipogenic or chondrogenic media to evaluate their multilineage differentiation potential. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Spinal ligaments were processed for immunostaining using MSC-related antibodies. Cells from human spinal ligaments could be extensively expanded with limited senescence. They were able to differentiate into osteogenic, adipogenic or chondrogenic cells. Flow cytometry revealed that their phenotypic characteristics met the minimum criteria of MSCs. Immunohistochemistry revealed the localization of CD90-positive cells in the collagenous matrix of the ligament, and in adjacent small blood vessels. We isolated and expanded MSCs from human spinal ligaments and demonstrated localization of MSCs in spinal ligaments. These cells may play an indispensable role in elucidating the pathogenesis of numerous spinal diseases.     

Functional identification of three receptor activator of NF-kappa B cytoplasmic motifs mediating osteoclast differentiation and function

Receptor activator of NF-kappa B ligand (RANKL) and its receptor activator of NF-kappa B (RANK) play pivotal roles in osteoclast differentiation and function. However, the structural determinants of the RANK that mediate osteoclast formation and function have not been definitively identified. To address this issue, we developed a chimeric receptor approach that permits a structure/function study of the RANK cytoplasmic domain in osteoclasts. Using this approach, we examined the role of six RANK putative tumor necrosis factor receptor-associated factor-binding motifs (PTM) (PTM1, ILLMT-REE(286-293); PTM2, PSQPS(349-353); PTM3, PFQEP(369-373); PTM4, VYVSQTSQE(537-545); PTM5, PVQEET(559-564); and PTM6, PVQEQG(604-609)) in osteoclast formation and function. Our data revealed that the RANK cytoplasmic domain possesses three functional motifs (PFQEP(369-373), PVQEET(559-564), and PVQEQG(604-609)) capable of mediating osteoclast formation and function. Moreover, we demonstrated that these motifs play distinct roles in activating intracellular signaling. PFQEP(369-373) initiates NF-kappa B, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 signaling pathways and PVQEET(559-564) activates NF-kappa B and p38 pathways in osteoclasts, whereas PVQEQG(604-609) is only capable of activating NF-kappa B pathway. Significantly, the revelation of these functional RANK cytoplasmic motifs has not only laid a foundation for further delineating RANK signaling pathways in osteoclasts, but, more importantly, these RANK motifs themselves represent potential therapeutic targets for bone disorders such as osteoporosis.     

Influence of ammonium chloride feeding time and light intensity on the cultivation of Spirulina (Arthrospira) platensis

This study dealt with the influence of both the feeding time and light intensity on the fed-batch culture of the cyanobacterium Spirulina (Arthrospira) platensis using ammonium chloride as a nitrogen source. For this purpose, a 2(2) plus star central composite experimental design combined with response surface methodology was employed, and the maximum cell concentration (X(m)), the cell productivity (P(X)), and the yield of biomass on nitrogen (Y(X/N)) were selected as the response variables. The optimum values of X(m) (1,833 mg L(-1)) and Y(X/N) (5.9 g g(-1)) estimated by the model at light intensity of 13 klux and feeding time of 17.2 days were very close to those obtained experimentally under these conditions (X(m) = 1,771 +/- 41 mg L(-1); Y(X/N) = 5.7 +/- 0.17 g g(-1)). The cell productivity was a decreasing function of the ammonium chloride feeding time and a quadratic function of the light intensity. The protein and lipid contents of dry biomass collected at the end of cultivations were shown to decrease with increasing light intensity.     

Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source

Arthrospira (Spirulina) platensis (Nordstedt) Gomont was autotrophically cultivated for biomass production in repeated fed-batch process using urea as nitrogen source, with the aim of making large-scale production easier, increasing cell productivity and then reducing the production costs. It was investigated the influence of the ratio of renewed volume to total volume (R), the urea feeding time (t_f) and the number of successive repeated fed-batch cycles on the maximum cell concentration (X_m), cell productivity (P_x), nitrogen-to-cell conversion yield (Y_x/n), maximum specific growth rate (μ_m) and protein content of dry biomass. The experimental results demonstrated that R = 0.80 and t_f = 6 d were the best cultivation conditions, being able to simultaneously ensure, throughout the three fed-batch cycles, the highest average values of three of the five responses (X_m = 2101 ± 113 mg L^?1, P_x = 219 ± 13 mg L^?1 d^?1 and Y_x/n = 10.3 ± 0.8 g g^?1).