Differential responses of nitrifying archaea and bacteria to methylene blue toxicity.

UNLABELLED: Methylene blue, a heterocyclic aromatic chemical compound used to treat fish diseases in the ornamental fish aquaculture industry, is believed to impair nitrification as a side effect. However, very little is known about the toxicity of methylene blue to nitrifying micro-organisms. Here, we report the susceptibility of six bacterial and one archaeal ammonia-oxidizing micro-organisms to methylene blue within the range of 10 ppb to 10 ppm. Remarkably high susceptibility was observed in the archaeal species Nitrosopumilus maritimus compared to the bacterial species. Ammonia oxidation by Nitrosopumilus maritimus was inhibited 65% by 10 ppb of methylene blue. Of the bacterial species examined, Nitrosococcus oceani was the most resistant to methylene blue toxicity. For similar inhibition of Nitrosococcus oceani (75% inhibition), one thousand times more methylene blue (10 ppm) was needed. The examination of single cell viability on Nitrosomonas marina demonstrated that methylene blue is lethal to the cells rather than reducing their single cell ammonia oxidation activity. The level of susceptibility to methylene blue was related to the cell volume, intracytoplasmic membrane arrangement and the evolutionary lineage of nitrifying micro-organisms. Our findings are relevant for effectively using methylene blue in various aquaculture settings by helping minimize its impact on nitrifiers during the treatment of fish diseases. In the future, resistant nitrifiers such as Nitrosococcus oceani may be purposely added to aquaculture systems to maintain nitrification activity during treatments with methylene blue. SIGNIFICANCE AND IMPACT OF THE STUDY: The susceptibility of six bacterial and one archaeal nitrifying micro-organisms to methylene blue was tested. Remarkably high susceptibility was observed in the archaeal species compared to the bacterial species. The level of resistance to methylene blue was related to the cell volume, cytomembrane system and the taxonomic position of the nitrifying micro-organisms. This may be significant in the design and management of engineered nitrification systems and the stability of the nitrification process in various ecosystems if these systems are exposed to harmful chemicals or toxins.

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea.

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Effect of reaction pH and CuSO4 addition on the formation of catechinone due to oxidation of (+)-catechin.

A novel hair dyeing technique being milder and safer for a human body is desired. The oxidation product of (+)-catechin, catechinone, was invented as a safer dyestuff for hair colouring under such the situation. The preparation of catechinone by a chemical oxidation is a practical way and the objective of the study is clarify the effect of the solution pH and in the presence or absence of Cu(2+) on the formation rate and yield of catechinone in order to improve the efficiency of the dye formation. The catechinone formation was monitored by ultraviolet-visible spectroscopy. Catechinone was prepared chemically from (+)-catechin in aqueous solution with O2 gas introduced over a pH range of 7.1-11.7. The rate and amount of the dye formation increase with increasing pH. Dissociation of the hydroxyl group of the catechol part of (+)-catechin is significant for the oxidation of (+)-catechin and promotes the dye production. This is because the deprotonated (+)-catechin has a higher reactivity with O2 . The production of catechinone is accelerated by the addition of CuSO4 and the production rate reaches the maximum at pH = 8.8. (+)-Catechin - Cu(2+) complexes are formed and the formation promotes the oxidation of the catechol part of (+)-catechin at pH ≤ 8.8. On the other hand, the complex becomes too stable to proceed for the oxidation reaction at pH > 8.8.

Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo.

BACKGROUND: Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma. METHODS: We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS). RESULTS: In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 mM). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU. CONCLUSION: These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

Characterization of monooxygenase gene diversity in benzene-amended soils.

AIM: To understand soil benzene monooxygenase gene diversity by clone library construction and microarray profiling. METHODS AND RESULTS: A primer set was designed, and benzene monooxygenase gene diversity was characterized in two benzene-amended soils. The dominant sequence types in the clone libraries were distinct between the two soils, and both sequences were assigned to novel clusters. Monooxygenase gene richness and diversity increased after benzene degradation. Oligonucleotide probes for microarray analysis were designed to detect a number of sequenced clones and reported monooxygenase genes. The microarray detected several genes that were not detected in the clone libraries of the same samples. Six probes were detected in more than one soil. CONCLUSIONS: The primer set designed in this study successfully detected diverse benzene monooxygenase genes. The level of diversity may have increased because the degradation of benzene differed from soil to soil. Microarrays have great potential in the comprehensive detection of gene richness as well as the elucidation of key genes for degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduces a new primer set that may be used to identify diverse benzene monooxygenase genes in the environment; moreover, it demonstrates the potential of microarray technology in the profiling of environmental samples.

Function of surfactants in hair dyeing by oxidation dyes 2. Effect on formation of oxidation dyes by p-aminophenol and 5-amino-o-cresol in dye bath(1).

The effect of surfactants on an oxidation-hair-dye-formation reaction in a dye bath was studied in order to learn the mechanism of the effect of surfactants on the dyeability of hair by the oxidation dye. The dye-formation behaviours for the p-aminophenol and 5-amino-o-cresol system with the surfactants, of which the hydrophilic parts have different charges, were compared changing the concentration of surfactants. It was found that the same dyes are produced, regardless of the charge of surfactants added, and the rate of dye produced in the dyebath is increased in the presence of surfactants. The order of the production rate is, with an anionic surfactant > with non-ionic surfactant > with cationic surfactant > without surfactant. The relation between the dyeability of hair and the rate of dye produced in the dyebath in the presence of surfactants is not found. The major factor governing the dyeability of hair is different from the mechanism of the increased dye in the solution. It was also found that the dye-formation rate is increased by immersing hair into the reaction solution, and hair works as an accelerator for the dye-formation reaction.

Micellar structure of beta-casein observed by small-angle X-ray scattering.

The small-angle X-ray scattering was observed from beta-casein micelles in 0.2 M phosphate buffer (pH 6.7) with varying temperatures. An oblate ellipsoid of a rigid core with a thin soft layer was proposed as a probable model of the beta-casein micellar structure, according to the results of the model optimization with simple triaxial bodies. Here the axial ratio was found to decrease and the micelle to become spherical when the polymerization proceeds with temperature. The consistency of the present model was examined with the results of hydrodynamic measurements published previously.

Self-assembling process of cylindrical virus coat proteins as observed by synchrotron small-angle X-ray scattering.

The self-assembly process of cucumber green mottle mosaic virus (CGMMV) protein and tobacco mosaic virus (TMV) protein was examined by the thermodynamic analysis of small-angle X-ray scattering (SAXS) data. Each polymerization step of the coat proteins was assumed to be specified by a single equilibrium constant, and the equilibrium constant was evaluated by fitting the size and shape of the constituents observed by SAXS to those calculated from an assumed polymerization scheme. The logarithmic plots of the equilibrium constant against the inverse of temperature were fitted with a straight line at each buffer concentration and the thermodynamic quantities were evaluated from its intercept (yielding entropy) and slope (yielding enthalpy). The enthalpy and entropy values of TMV protein were found to be independent of buffer concentration, whereas those of CGMMV protein depended strongly on buffer concentration. In the limit, as ionic strength tends to infinity, both the enthalpy and entropy values of CGMMV protein approach those of TMV protein. The higher negative surface charge of CGMMV protein is considered to be responsible for the formation of stable single-layered disks, and for the slow polymerization process even at higher temperature and higher buffer concentrations.

16S rDNA genotyping using PCR/RFLP (restriction fragment length polymorphism) analysis among the family Vibrionaceae.

The 16S rDNA genotypes among the family Vibrionaceae were determined using PCR/RFLP analysis. Five tetrameric restriction enzymes (HhaI, DdeI, RsaI, Sau3AI and MspI) were used for RFLP analysis and adequate numbers of informative bands were obtained from each enzyme. Twenty-seven genotypes were obtained from 49 type and reference strains including 35 species. Nineteen species could be assigned to specific 16S rDNA genotypes, supporting the application of this analysis for identification. Trees constructed using five endonucleases resolved groups almost identical to those inferred from 16S rRNA gene sequencing. However, the branch lengths and detailed relationships among strains within a group differed from those inferred from sequence comparisons. The results of this study should be useful for genotyping, identification and approximate classification of natural isolates belonging to the family Vibrionaceae.

A proposal to transfer Vibrio marinus (Russell 1891) to a new genus Moritella gen. nov. as Moritella marina comb. nov.

The taxonomic positions of Vibrio marinus and 11 related natural isolates were examined. Their phylogenetic positions on the basis of almost complete 16S rRNA sequences were determined by maximum likelihood, maximum parsimony and neighbor-joining analyses. V. marinus and the 11 isolates fell into a single cluster that was clearly distinct from other genera. There is now strong evidence that V. marinus should be reclassified as Moritella marina gen. nov., comb. nov.

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching.

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.

Characterization of psychrotrophic bacteria in the surface and deep-sea waters from the northwestern Pacific Ocean based on 16S ribosomal DNA analysis.

Seventy-eight 4 degrees C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0-200 m) and deep-sea (1000-9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4 degrees C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats.

Conformation of cyclic and linear (1 --> 2)-beta-D-glucans in aqueous solution.

The conformations of cyclic (1 --> 2)-beta-D-glucan chains having degree of polymerization (dp) 17 to 24 were characterized by means of small-angle X-ray scattering and Monte Carlo simulation. The results indicate that cyclic (1 --> 2)-beta-D-glucan chains adopt the shape of a doughnut-like ring with a thickness of about 10 A for all the samples. The diameter of the annulus for the cyclic glucan having dp 21 is estimated to be only about 4-5 A. Two linear (1 --> 2)-beta-D-glucans possessing dp 19 and 21 prepared by acid hydrolysis of a cyclic glucan and subsequent fractionation showed different scattering profiles from those obtained for cyclic glucans having the corresponding dp. Although the Monte Carlo simulation does not completely reproduce the scattering profiles observed by small-angle X-ray scattering, linear (1 --> 2)-beta-D-glucans seem to possess a characteristic cylindrical shape with cross-sectional diameters of 11.8 and 13.2 A for linear glucans of dp 19 and 21, respectively.

Low-degree oxidized scleroglucan and its hydrogel.

A controlled oxidation of scleroglucan was performed with sodium periodate to prepare aldehyde derivatives (scleraldehyde) with a low degree of oxidation (10 and 20%), which were utilized for crosslinking reactions with hexamethylenediamine. The structural characterization of scleraldehydes and their corresponding hydrogels was attempted by small-angle X-ray scattering (SAXS). While scleraldehyde with a higher degree of oxidation (> or = 50%), according to an earlier research, was found to disentangle into single chains as the degree of oxidation increases; scleroglucan bearing a low percentage of aldehydic groups (up to 20%) retains mainly the conformation of the natural polysaccharide, thus the system can be represented as composed of triple helices with only minor disentanglements at the sites where the aldehyde groups are present. The hydrogel prepared from scleraldehyde with a low degree of oxidation is brittle and fragmented, in contrast to the elastic/homogeneous hydrogel earlier prepared from scleraldehyde with a high degree of oxidation. The hydrogel from scleraldehyde with a low degree of oxidation was found to possess a network structure that consisted mostly of the triple helices crosslinked in specific points where the triple helices are disentangled into single chains because of the presence of the aldehyde groups.

Analysis of the origin of the right inferior phrenic artery in 178 patients with hepatocellular carcinoma treated by chemoembolization via the right inferior phrenic artery.

BACKGROUND: No previous report has described the level of the origin of the right inferior phrenic artery (RIPA) based on an analysis of the relationships between the level of the RIPA, the celiac artery (CA), the superior mesenteric artery (SMA), and the right renal artery (RRA) in a series of cases. PURPOSE: To evaluate the origin of the RIPA by retrospectively analyzing angiographic findings in 178 patients with hepatocellular carcinoma (HCC) who underwent transcatheter arterial chemoembolization (TACE) via the RIPA. MATERIAL AND METHODS: In patients treated with intraarterial chemoembolization for HCC, additional superselective chemoembolization of the RIPA branches was necessary in 178 cases. We analyzed the level of the origin of the RIPA in these patients according to the relationships between the level of the origin of the RIPA, the CA, the SMA, and the RRA on angiography. RESULTS: Among the 178 cases, the RIPA arose from 1) the aorta directly in 102 cases (57%), 2) the CA in 53 (30%), 3) the left gastric artery (LGA) in three (2%), 4) the dorsal pancreatic artery (DPA) in one (1%), and 5) the RRA in 19 (11%). The level of the origin of the RIPA that originated directly from the aorta was supraceliac in 56 cases (32%), between the CA and the SMA in 31 (17%), and between the SMA and the RRA in 15 (8%). CONCLUSION: In our study, the RIPA originated from the aorta between the CA and the SMA directly in 17% of cases. When it is difficult to identify the origin of the RIPA, we must keep in mind that the RIPA may originate from the right part of the aorta within the small distance between the SMA and the CA.

Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling.

Depth-related changes in whole-community structure were evaluated in a coastal marine sediment using a molecular fingerprinting method, terminal restriction fragment length polymorphism (T-RFLP) analysis, and a chemotaxonomic technique (quinone profiling). Dendrograms derived from both T-RFLP analysis and quinone profiling indicated a significant variation in microbial community structure between the 0-2 cm layer and deeper layers. This corresponded to the dramatic change in the redox potential, acid-volatile sulphide-sulphur and bacterial numbers observed at 0-2 cm and 2-4 cm depths. A significant change in the number of terminal restriction fragments (T-RFs) was also detected at this transition depth. However, the change in major T-RFs with depth was not seen in electropherograms. The population changes were primarily variations in minor ribotypes. Most quinone homologues were detected at all depths, although the quinone composition changed with depth. Therefore, quinone profiling also suggested that the depth-related variation was primarily attributable to minor bacterial groups rather than change in the major population structure. 16S rDNA clone library analysis revealed that clones belonging to the genera Vibrio and Serratia predominated as major bacterial groups at all depths. Our data suggested that the sediment community might result from sedimentation effects of sinking particles. Overall, our results demonstrated that the combined methods of T-RFLP analysis and quinone profiling were effective for assessing depth-related microbial populations.

A new approach to separate the genus Photobacterium from Vibrio with RFLP patterns by HhaI digestion of PCR-amplified 16S rDNA.

A new approach to separate members of the genus Photobacterium from the genus Vibrio with RFLP (Restriction Fragment Length Polymorphism) patterns by HhaI digestion of PCR-amplified 16S rDNA was developed in the present study. It was clearly shown that these patterns of the genus Photobacterium were unique and distinguishable from Vibrio species. This method is very simple and does not need other supporting procedures, such as Southern transfer and probe hybridization. It can be applied not only to luminous species, but also to non-luminous Photobacterium spp. This result promises a rapid tool to distinguish the genus Photobacterium from Vibrio and should be useful in routine identification system.

Facile synthesis of Nalpha-protected-L-alpha,gamma-diaminobutyric acids mediated by polymer-supported hypervalent iodine reagent in water.

Hofmann rearrangement of Nalpha-Boc-L-Gln-OH mediated by a polymer-supported hypervalent iodine reagent poly[(4-diacetoxyiodo)styrene] (PSDIB) in water afforded Nalpha-Boc-L-alpha,gamma-diaminobutyric acid (Boc-Dab-OH, 1) in 87% yield. Nalpha-Z-derivative (Z-Dab-OH, 2) was prepared with PSDIB in 83% yield. Since the reaction of Nalpha-Fmoc-Gln-OH by this procedure did not proceed because of the insolubility of Fmoc-Gln-OH in aqueous media, we synthesized Fmoc-Dab(Boc)-OH (5) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution-phase synthesis.

Reassessment of the taxonomic position of Vibrio iliopiscarius (Onarheim et al. 1994) and proposal for Photobacterium iliopiscarium comb. nov.

The phylogenetic position of Vibrio iliopiscarius was inferred by the maximum-likelihood, maximum-parsimony and neighbour-joining methods on the basis of almost complete 16S rRNA gene sequences. The results showed that this species falls into the same cluster as Photobacterium species and is clearly distinct from other Vibrio species. Its nearest phylogenetic neighbour is Photobacterium phosphoreum. From these results, it is concluded that V. iliopiscarius should be reclassified as Photobacterium iliopiscarium comb. nov., the type strain of which is PS1T (= ATCC 51760T).

16S rDNA restriction fragment length polymorphism analysis of psychrotrophic vibrios from Japanese coastal water.

Restriction fragment length polymorphism (RFLP) analysis was carried out for 136 natural isolates belonging to the family Vibrionaceae. These were collected from inshore areas of Japan, mainly in winter. Twenty-eight 16S rDNA genotypes were obtained by digestion with four restriction endonucleases (HhaI, DdeI, RsaI, and Sau3AI). To estimate the genetic relationships, 53 informative fragments were scored by their presence or absence. A dendrogram was constructed using the unweighted pair group method with the arithmetic averages algorithm. Five RFLP groups (groups I to V) were obtained. Group I corresponded to Vibrio splendidus-like strains. It was confirmed that this group was not only found in Otsuchi Bay, but also in broad coastal areas of Japan. Group II strains were not identified as previously known Vibrio species. Group III strains were regarded as members of the Vibrio main group, which is a major phylogenetic group deduced from 16S rRNA gene analysis in the family Vibrionaceae. The RFLP profile indicated that Group IV strains were closely related to V. hollisae. Group V strains showed RFLP patterns which have not been observed previously. From the clustering analysis, it was concluded that group V strains were not Vibrio species. Most of the isolates studied were not identified as previously described species. It suggests that many psychrotrophic vibrios in cold marine environments remain as unknown species.