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A Nonomuraea sp. strain MM565M-173N2 was isolated from deep-sea sediment off the Sanriku coast, and new antibiotics were evaluated against carbapenem-resistant Enterobacteriaceae (CRE), which is a problematic group of bacteria because of their antimicrobial resistance. From 220 l of fermented broth from strain MM565M-173N2, we isolated four new antibiotics by gel filtration and HPLC, designated as sealutomicins A (1.8 mg), B (1.5 mg), C (0.8 mg), and D (0.8 mg). Their structures were determined from MS, NMR, and CD spectra. Sealutomicin A was found to be a new enediyne antibiotic, while sealutomicins B-D were aromatized products from sealutomicin A. Sealutomicin A showed strong antibacterial activity (MIC 0.05-0.2 μg ml-1) against CRE.

Introduction Quantitative measurement of anti-A/-B antibody titers is important during ABO-incompatible living kidney transplantation (ABOi-LKT). Methods We conducted a multi-institutional study to measure the antibody titers using the automated column agglutination technique (auto-CAT) and tube test (TT) method in ABOi-LKT recipients. Statistical analysis was performed to evaluate the two methods. Results We examined 111 samples from 35 ABOi-LKT recipients at four institutions. The correlation coefficient of the two methods was >0.9; the concordance rate and clinically acceptable concordance rate for the IgG titers were 60.4% and 88.3%, respectively. Perioperative status did not influence the statistical significance. Parallel changes were observed in the IgG antibody titers measured using the auto-CAT or TT technique by desensitizing therapy in time-course monitoring. Conclusion Auto-CAT is comparable with the TT technique and is feasible for IgG anti-A/B antibody titration in ABOi-LKT recipients. This article is protected by copyright. All rights reserved.

The present study addresses the microbiome of the first whale fall (YOKO 16) that has been described in the deep sea in the southern Atlantic Ocean (Sao Paulo Plateau; 4204 m depth), in terms of its metabolic uniqueness. Sets of ten thousand protein sequences from YOKO 16 and 29 public domain metagenomes (SRA and GenBank databases) that represent various marine, terrestrial and gut-associated microbial communities were analyzed. The determination of protein functionality, based on the KAAS server, indicated that the YOKO 16 microbiome has industrially-relevant proteins, such as proteases and lipases, that have low similarity (~50%) with previously-described enzymes. The amino acid usage in the YOKO 16 protein sequences (based on blastp and Clustal analysis) revealed a pattern of preference similar to that of extremophiles, with an increased usage of polar, charged and acidic amino acids and a decreased usage of nonpolar residues. We concluded that the targeted microbiome is of potential biotechnological use, which justifies the allocation of resources for the discovery of enzymes in deep-sea whale fall communities.

We have isolated several poly 3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBH) degradable bacteria from the deep-sea sediment samples. The isolated microbes were involved into α-, γ- Proteobacteria g...

We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10–60 °C and 0.1–350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10–60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1–200 MPa, but the specific activity ratios (1.28–1.59) of 10C9/wild-type enzyme at 50–200 MPa at 30 °C were smaller than those at 10–60 °C (1.73–4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the β-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the β-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.

Identification of specific drug targets is very important for cancer therapy. We recently identified zinc finger protein of the cerebellum 5 (ZIC5) as a factor that promotes melanoma aggressiveness by platelet-derived growth factor D (PDGFD) expression. However, its roles in other cancer types remain largely unknown. Here we determined the roles of ZIC5 in prostate cancer (PCa) and colorectal cancer (CRC) cells. Results showed that ZIC5 was highly expressed in CRC and dedifferentiated PCa tissues, whereas little expression was observed in relevant normal tissues. Knockdown of ZIC5 decreased proliferation of several PCa and CRC cell lines with induction of cell death. ZIC5 knockdown significantly suppressed PDGFD expression transcriptionally, and PDGFD suppression also decreased proliferation of PCa and CRC cell lines. In addition, suppression of ZIC5 or PDGFD expression decreased levels of phosphorylated focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) which are associated with PCa and CRC aggressiveness. Furthermore, knockdown of ZIC5 or PDGFD enhanced death of PCa and CRC cells induced by the anti-cancer drugs docetaxel or oxaliplatin, respectively. These results suggest that ZIC5 and PDGFD promote survival of PCa and CRC cells by enhancing FAK and STAT3 activity, and that the roles of ZIC5 are consistent across several cancer types.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Fifteen actinomycetes were isolated from deep-sea sediments in Calyptogena communities in the Japan Trench (depth: 5352 m) and Sagami Bay (depth: 1171 m) under incubation at 4–37 °C. A relatively large number of colonies were obtained with incubation at 30 °C from the deep-sea sediment of Sagami Bay. We found Rhodococcus, Nocardioides and Micromonospora actinomycetes in both sediments, and additionally isolated an Aeromicrobium actinomycete from the Japan Trench and Streptomyces and Actinomadura actinomycetes from Sagami Bay. Actinomadura sp. DS-MS-114 isolated from the deep-sea sediment in Sagami Bay produced an antibacterial compound against Staphylococcus aureus NBRC12732T. Structural analysis by liquid chromatography–high-resolution electrospray ionization mass spectrometry (LC-HRESIMS) and proton nuclear magnetic resonance (1H-NMR) indicated that the antibacterial compound was 5,6-dihydro-1,8-dihydroxy-3-methylbenz[a]anthracene-7,12-quinone, which is known as an immunosuppressant. The deep-sediment in the Calyptogena community may be useful as a source of various actinomycetes producing physiologically active substances.

New bioactive substances were identified from several marine actinomycetes strains by LC-HRESI-MS based non-targeted metabolomics. A new siderophore and its derivative, named fradiamines A and B, were isolated from the extract of the deep-sea actinomycetes Streptomyces fradiae MM456M-mF7 by Diaion CHP-20P, Sephadex LH-20 column chromatography and HPLC. Fradiamine A was a new compound, but fradiamine B was previously patented as a sweetness enhancer. Their structures were determined by NMR and LC-HRESI-MS/MS analysis. Fradiamines A and B contained two alkyl amines asymmetrically bonded to citrate, a type of structure derived from actinomycetes and other bacteria and rarely observed in siderophores. Fradiamines A and B showed moderate antibiotic activity against Clostridium difficile with IC50 values of 32 and 8 μg ml-1, respectively.

Shewanella benthica DB21MT-2 is an obligate piezophilic bacterium isolated from the sediment of the Mariana Trench at depth of 10,898 m. Here we report the complete genome sequence of S. benthica DB21MT-2, which consists of a single circular chromosome with the size of 4,353,505 bp and G + C content of 45.86%. Comparative genomic analysis reveals the strain possesses multiple pathways for energy generation as has been reported in other deep-sea bacteria. In addition, several genes predicted to be involved in flagellar synthesis or component of toxin-antitoxin system are specifically found in obligate piezophilic strains from hadal trench. The result is expected to provide novel information for a better understanding on bacterial obligate piezophilic life-style and on bacterial adaptation to hadal trench environment.

Vestimentiferan tubeworms acquire their symbionts through horizontal transmission from the surrounding environment. In the present study, we constructed a 16S rRNA gene clone library to investigate the phylogenetic relationship between diverse microbes in the sediment and symbiotic bacteria in the trophosome of the tubeworm, Lamellibrachia satsuma, from Kagoshima Bay, Japan. Two symbiotic bacterial phylotypes belonging to the classes γ- and e-Proteobacteria were found from this tubeworm trophosome. They were very closely related to the symbionts of several other marine invertebrates. The most predominant bacteria in the sediment were e-Proteobacteria. A broad diversity of bacteria belonged to non-proteobacterial phyla such as Planctomycetes, Acidobacteria, and Chloroflexi was observed. The presence of sulfur oxidizers (i.e., e-Proteobacteria and γ-Proteobacteria) and sulfur reducers (i.e., δ-Proteobacteria) may play a significant role in the sulfur cycle in these habitats and provide multiple sources of nutrition to the cold-seep communities. Closely related clones of e-Proteobacteria symbiont in the species level and of γ-Proteobacteria symbiont in the genus level were found in the surrounding sediment. The similarity of symbiont clones of L. satsuma with other symbionts and free-living bacteria suggests the possibility of opportunistic symbiosis in e-Proteobacteria and the co-evolution of γ-Proteobacteria having occurred after symbiosis with the tubeworms.

Licensing by self MHC class I ligands is required for proper natural killer (NK) cell response. NK cells with inhibitory killer cell immunoglobulin-like receptors for nonself MHC exhibit transient alloreactivity after hematopoietic stem cell transplantation (HSCT). We analyzed 3866 recipients in the Japan national registry who underwent their first allogeneic HSCT for acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) from HLA-A, -B, and -DRB1 allele-genomatched unrelated donors. By classifying them into 5 independent groups based on HLA-C group matching and assumed donor NK cell status, we found that for HLA-C–matched HSCT for AML in HLA-C1/C1 recipients, in whom transient alloreactivity against HLA-C2–negative leukemic cells was expected, the relapse rate was significantly lower than it was in HLA-C–matched HSCT for AML in HLA-C1/C2 recipients (hazard ratio [HR], .72; P = .011). This difference was not observed in HLA-C–matched HSCT for ALL. Compared with HLA-C–matched HSCT, significantly higher mortality was observed in HLA-C1/C1 AML patients who received transplants from HLA-C–mismatched HLA-C1/C1 donors (HR, 1.37; P = .001) and in HLA-C1/C1 ALL patients who received transplants from HLA-C2–positive donors (HR, 2.13; P = .005). In conclusion, donor selection based on leukemic subtype and donor HLA-C group matching improves transplantation outcome after HLA-C–mismatched HSCT.

Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation. Deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.

The deep-sea denitrifier Pseudomonas sp. MT-1 has two gene clusters encoding dissimilatory nitrate reductases, periplasmic nitrate reductase (Nap) and membrane-bound nitrate reductase (Nar). In order to investigate the physiological role of these enzymes, we constructed the disrupted mutants of napA, narG, and narK (encoding the catalytic subunits of Nap and Nar, as well as the nitrate transporter, respectively). The napA mutant showed almost the same growth rate as the wild-type under both atmospheric and high pressure of 30 MPa. On the other hand, the narG and narK mutants showed growth deficiencies under atmospheric pressure which were more pronounced at a pressure of 30 MPa. Thus, Nar was shown to be the dominant dissimilatory nitrate reductase in MT-1, especially under high pressure, whereas Nap can support the growth with denitrification to some extent. Further, nitrate reductase activity of the soluble and membrane fractions of MT-1 was measured under high pressure. Both activities were highly piezotolerant even under a pressure of 150 MPa. Therefore, the stability of nitrate reductases under high pressure is not a limiting step for the growth of MT-1 under these conditions. Although the reason why Nar rather than Nap is dominant and the physiological role of Nap in MT-1 are still unclear, we have demonstrated the mechanisms of the denitrification system in the environment of the deep-sea.

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4–70 °C and pH 2.0–12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.

Here, we report the genome sequence of Bacillus subtilis strain 29R7-12, a piezophilic bacterium isolated from coal-bearing sediment down to ~2.4 km below the ocean floor in the northwestern Pacific. The strain is a Gram-positive spore-forming bacterium, closely related to Bacillus subtilis within the phylum Firmicutes . This is the first complete genome sequence of a Bacillus subtilis strain from the deep biosphere. The genome sequence will provide a valuable resource for comparative studies of microorganisms from the surface and subsurface environments.

Marinilactibacillus piezotolerans strain 15R is a facultatively anaerobic heterotrophic lactobacillus isolated from deep marine subsurface sediment nearly 2 km below the seafloor in the northwestern Pacific. We report here the first whole-genome sequence of strain 15R. The identified genome sequence has 2,767,908 bp, 35.4% G+C content, and predicted 2,552 candidate protein-coding sequences, with no identified plasmids.

Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.

Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.

The deep ocean piezosphere accounts for a significant part of the global ocean, hosts active and diverse microbial communities which probably play a more important role than hitherto recognized in the global ocean carbon cycle. The conventional biological pump concept and the recently proposed microbial carbon pump mechanism provide a foundation for our understanding of the role of microorganisms in cycling of carbon in the ocean. However, there are significant gaps in our knowledge and a lack of mechanistic understanding of the processes of microbially-mediated production, transformation, degradation, and export of marine dissolved and particulate organic matter (DOM and POM) in the deep ocean and the ecological consequence. Here we propose the POM-DOM piezophilic microorganism continuum (PDPMC) conceptual model, to address these important biogeochemical processes in the deep ocean. We propose that piezophilic microorganisms (bacteria and archaea) play a pivotal role in deep ocean carbon cycle where microbial production of exoenzymes, enzymatic breakdown of DOM and transformation of POM fuels the rapid cycling of marine organic matter, and serve as the primary driver for carbon cycle in the deep ocean.

The δD of fatty acids is emerging as an important marine biogeochemical proxy, but the microbiological and environmental factors controlling the variations of δD of the lipids are not fully constrained. We report here the first measurement of D/H ratios of fatty acids in a piezophilic bacterium and show that hydrostatic pressure and the lipid biosynthetic pathway probably exerts dominant control over the δD of fatty acids. Piezophilic bacterium Moritella japonica DSK1 was grown at a pressure of 30 MPa with glucose as substrate. Fatty acids in DSK1 showed vastly varied δD, ranging from + 44.4 to − 171‰. Short-chain fatty acids (SCFA), which are synthesized by the fatty acid synthase (FAS) pathway, had positive δD (average + 3‰), whereas long-chain polyunsaturated fatty acid (LC-PUFA) synthesized via the polyketide pathway exhibited much depleted δD (− 171‰). Our results suggest that the lipid biosynthetic pathways can exert first-order control on the hydrogen isotope signature of bacterial membrane lipids under elevated pressure. Our findings have important implications in marine biogeochemistry. D-depleted fatty acids in marine sediments and in the water column may be derived from piezophilic bacterial reworking and resynthesis of organic matter at high pressure condition. Thus, caution must be exercised in the interpretation of hydrogen isotope signatures of lipids in, e.g., deducing sources of organic matter and tracing microbial biogeochemical processes in the deep ocean and the deep biosphere.

We developed an electrical modulation method for attachment and detachment of microorganisms. Living microorganisms suspended in non-nutritive media such as PBS(−) and artificial seawater were attracted by and selectively attached to indium tin oxide (ITO)/glass electrode regions to which a negative potential was applied. The microorganisms suspended in LB medium and glucose solution were not attracted to the ITO electrode. Dead microorganisms were not attracted to the ITO electrode. The living microorganisms were retrieved after detachment from the ITO electrode by application of a high-frequency triangular wave potential. When we applied this method to separate microorganisms from deep-sea sediment, bacteria belonging to 19 phyla and 23 classes were collected without undesirable high molecular weight contaminants such as humic acids. At the phylum and class level, respectively, 95 and 87 % of the phylotypes among electrically retrieved bacteria were common to the gene clones from the direct sediment DNA extraction. This technique is a novel useful method to prepare bacterial cells in a single population or a community for metagenomic analyses.

We have developed an efficient method of screening to detect pressure-sensitive mutants of barophilic or barotolerant bacteria using conventional agar medium plates. By this new method, 75 colonies can be screened per plate under high hydrostatic pressure.

Insights into mechanisms of drug resistance could extend the efficacy of cancer therapy. To probe mechanisms in melanoma, we performed siRNA screening of genes that mediate the development of neural crest cells, from which melanocytes are derived. Here, we report the identification of ZIC5 as a mediator of melanoma drug resistance. ZIC5 is a transcriptional suppressor of E-cadherin expressed highly in human melanoma. ZIC5 enhanced melanoma cell proliferation, survival, drug resistance, in vivo growth and metastasis. Microarray analysis revealed that ZIC5 downstream signaling included PDGFD and FAK activation, which contributes to drug resistance by enhancing STAT3 activation. Silencing of ZIC5 or PDGFD enhanced the apoptotic effects of BRAF inhibition and blocked survival of melanoma cells resistant to BRAF inhibitors. Furthermore, inhibition of FAK or STAT3 suppressed expression of ZIC5, which was positively regulated by PDGFD, FAK, and STAT3 in a positive feedback loop. Taken together, our results identify ZIC5 and PDGFD as candidate therapeutic targets to overcome drug resistance in melanoma. Cancer Res; 77(2); 366–77. ©2016 AACR.

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery.

Organisms living in deep seas such as the Mariana Trench must be adapted to the extremely high pressure environment. For example, the 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 (SbIPMDH) remains active in extreme conditions under which that from the land bacterium S. oneidensis MR-1 (SoIPMDH) becomes inactivated. In order to unravel the differences between these two IPMDHs, their structures were determined at ~1.5 Å resolution. Comparison of the structures of the two enzymes shows that SbIPMDH is in a more open form and has a larger internal cavity volume than SoIPMDH at atmospheric pressure. This loosely packed structure of SbIPMDH could help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than SoIPMDH.

Structures of 3-­isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration., Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

For efficient oxygen supply to pressurized culture, we developed a method using a highly pressurized membrane reactor with an air-saturated medium circulation system. The new method increased the cell growth of aerobic yeast approximately 20 folds larger than that in the case of using a conventional method.

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia call (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53 ml/mol at 25 degrees C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

All the self-reproducing cellular organisms so far examined have DNA as the genome. However, a DNA-less organism carrying an RNA genome is suggested by the fact that many RNA viruses exist and the widespread view that an RNA world existed before the present DNA world. Such a possibility is most plausible in the microbial world where biological diversity is enormous and most organisms have not been identified. We have developed experimental methodology to search DNA-less microorganisms, which is based on cultivation with drugs that inhibit replication or expression of DNA, detection of DNA in colonies with a fluorescent dye and double staining for DNA and RNA at a cellular level. These methods have been applied for about 100 microbial samples from various waters including hot springs, soils including deep sea sediments, and organisms. We found many colonies and cells which apparently looked DNA-less and examined them further. So far, all such colonies that reformed colonies on isolation were identified to be DNA-positive. However, considering the difficulty in cultivation, we think it possible for DNA-less microorganisms to live around us. We believe that our ideas and results will be of interest and useful to discover one in the future.

To investigate biodegradability of so-called biodegradable plastics in deep sea, degrading behavior of aliphatic polyesters poly( ɛ -caprolactone) [PCL], poly( β -hydroxybutyrate/valerate) [PHB/V], and poly(butyrene succinate) [PBS] in deep seawaters were evaluated at Rausu, Toyama, and Kume, Japan. After 12 months of soaking in deep seawaters, PCL and PHB/V fibers became brittle or completely ruined. Although breaking strength of PBS fibers maintained approximately 95% of the initial values, for all 3 fibers, many pinholes and cracks were observed on the surfaces. These results imply that examined 3 aliphatic polyesters are degradable also at deep sea though the degradation rates would vary with the material structures. From the deep seawaters assessed, 5 PCL-degrading bacteria were isolated, which were found to belong to the genuses Pseudomonas , Alcanivorax , and Tenacibaculum , as assessed by phylogenetic analysis using 16S rRNA gene sequencing. It was also confirmed that all these bacteria degrade PCL fibers in vitro. In addition, these strains were found to prefer conditions of low temperature (4–10 °C) and high hydrostatic pressure.

Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.

In this study, we investigate the potential of peritoneal macrophages to differentiate into dendritic cell (DCs) in response to preferential uptake of oligomannose-coated liposomes (OMLs). About 30% of peritoneal cells (PECs) preferentially took up OMLs that were administered into the peritoneal cavity. The OML-ingesting cells expressed CD11b and F4/80, but lacked CD11c expression, indicating that the OML-ingesting PECs with a CD11b(high)CD11c(-) phenotype are resident peritoneal macrophages. During in vitro cultivation, CD11c(+) cells arose among the PECs with ingested OMLs. CD11c(+) cells also developed among enriched peritoneal CD11b(high)CD11(-) cells from OML-treated mice, and the resulting CD11c(+) cells expressed co-stimulatory molecules and MHC class II. In addition, OML-ingesting CD11b(high)CD11c(+) cells were found in spleen after the enriched peritoneal macrophages with ingested OMLs were transplanted in the peritoneal cavity of mice. These results show that a fraction of peritoneal macrophages can differentiate into mature DCs following uptake of OMLs.

We have isolated thirteen different bacterial strains as poly e-caprolactone (PCL)-degrading bacteria from the Kurile and Japan Trenches at a depth of 5,000-7,000 m (deeper ocean bottoms). The isolates belong to the Shewanella, Moritella, Psychrobacter and Pseudomonas genera. This is the first record of PCL degrading bacteria isolated from deep-sea environments at depth of over 5,000 m. Six strains of the isolates, numbered CT01 in genus Shewanella, CT12, JT01 and JT04 in genus Moritella, JT05 in genus Psychrobacter, and JT08 in genus Pseudomonas were selected for investigation of their cell shapes, degrading abilities for several aliphatic polyesters, and growth profiles. The cell shapes of the strains, except JT05, were rod-shaped, non-spore-forming and motile by means of a single or multi polar flagella. The cell shapes of JT05 were coccal with no visible flagella. From the results of degradation tests on six different alihphatic polyesters, all strains could degrade only PCL. Strains CT01, CT12, JT01 and JT04 are psychrophilic and pressure tolerant bacteria and three strains except JT04 showed typical piezophilic growth profiles. Therefore, it is possible that these strains might play a role in degrading aliphatic polyesters under deep-sea conditions, ie., low-temperatures and high hydrostatic pressures.

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74–90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K m and k cat; although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k cat/K m) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.

We obtained 19 individual DNA fragments encoding 2-deoxy-scyllo-inosose synthase involved in the biosynthesis of aminoglycoside antibiotics from deep-sea sediments of the Pacific Ocean. Compared with genes from land-based environmental DNA, they showed low diversity. Combined with our previous study concerning the discovery of other aminoglycoside-biosynthetic genes from the same deep-sea samples, we suggest the importance of exploration of multiple biosynthetic genes to determine the diversity of aminoglycoside producers. We found that the deep sea is a useful source for screening of these genes.

Arabidopsis response regulators (ARRs) are involved in the His-to-Asp phosphotransfer signal transduction mechanism induced by cytokinins, a class of phytohormones in Arabidopsis thaliana. Previous studies have reported ARR mRNA accumulation in response to cytokinins. ARRs consist of 2 families (types A and B). The expression of type-A ARR genes can be induced by cytokinins, while that of type-B genes is constitutive. Recently, we have shown that plant growth induced by the endocrine disruptor bisphenol A (4,4� -isopropylidenediphenol) was similar to that induced by cytokinins. The present study is the first to show that ARR (type-A family; ARR15 and ARR16) mRNA accumulation is influenced by bisphenol A, indicating that bisphenol A is involved in inducing intracellular signal transduction.

A procedure for the isolation of aliphatic polyester-degrading bacteria from deep-sea environments was developed using three step high-pressure cultivation. First, the sediments collected from the deep-sea floor, the Kurile and Japan Trenches at a depth of 5000-7000 m, were cultivated with poly e-caprolactone (PCL) films under low-temperature (4°C) and high-pressure (50MPa) conditions. Secondly, high-pressure continuous cultivation using the deep-sea baro-piezophile and thermophile isolation and cultivation (DEEPBATH) system was performed three times to accumulate piezophilic bacteria. Finally, piezophilic PCL degrading bacteria were isolated using the pressure bag method involving media with PCL granules. Using this procedure, we succeeded in isolating several piezophilic aliphatic polyester-degrading bacteria from the sediment samples of deep-trenches.

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

To understand sulfur oxidation in thioauto-trophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox)(soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-alpha, and the JNK inhibitor inhibited the antibody-induced TNF-alpha production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-alpha production in macrophage-like RAW264.7 cells.

We achieved the purification of three alpha-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for alpha-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the alpha- and beta-keto esters, but also toward alpha-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.

Resident peritoneal macrophages (PEMs) express SIGNR1 on the cell surface as a major mannose receptor. These cells also ingest oligomannose-coated liposomes (OMLs) in an oligomannose-dependent manner following intraperitoneal administration. Therefore, the current study was conducted to investigate the possible role of SIGNR1 in capture of OMLs. Transient expression of several SIGN-related lectins potentially expressed on PEMs in CHO cells revealed that only SIGNR1 contributed to capture of OMLs. When SIGNR1 was introduced into mouse macrophage-like RAW264.7 cells, SIGNR1-expressing RAW (RAW-SIGNR1) cells recognized OMLs under serum-free conditions. OML recognition by RAW-SIGNR1 cells as well as that by PEMs was partially inhibited by an anti-SIGNR1 antibody (ER-TR9) and by mannan, and completely inhibited by EDTA. Interestingly, OML recognition by RAW-SIGNR1 cells was accelerated in the presence of serum, partially inhibited by an anti-complement receptor 3 (CR3) antibody (M1/70), and almost completely inhibited by a combination of ER-TR9 and M1/70. Complete inhibition of OML ingestion by the combination of ER-TR9 and M1/70 was also observed under serum-free conditions, suggesting that SIGNR1 and CR3 cooperate in an additive way in capture of OMLs by macrophage-like RAW cells. Administration of ER-TR9 or M1/70 into the peritoneal cavity led to a significant decrease of OML uptake by PEMs. Therefore, SIGNR1 expressed on macrophages acts as a receptor for recognition of OMLs under physiological conditions.

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.

Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP. Results: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction. Conclusions: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

SummaryAlthough dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.