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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.

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.

Little is known regarding the chimerism status after reduced-intensity conditioning transplantation when bone marrow is used as a stem cell source. We prospectively analyzed lineage-specific chimerism and retrospectively evaluated clinical outcomes in 80 adult patients who underwent unrelated donor bone marrow transplantation (URBMT) with fludarabine plus melphalan (FM) as the conditioning regimen. Mixed donor chimerism (MDC) was seen in 43 and 10 % of patients at days 14 and 28, respectively. Melphalan at ≤130 mg/m(2) was associated with an increased incidence of MDC at day 28 (P = 0.03). Patients with MDC at day 14 showed a marginally increased risk of primary graft failure and a marginally decreased risk of graft-versus-host disease. In multivariate analysis, MDC at day 14 was associated with higher overall mortality (hazard ratio (HR) = 2.1; 95 % confidence interval (CI), 1.1-4.2; P = 0.04) and relapse rate (HR = 3.0; 95 % CI, 1.2-7.5; P = 0.02), but not with non-relapse mortality (HR = 1.8; 95 % CI, 0.70-4.6; P = 0.23). Thus, the FM regimen yields prompt complete donor chimerism after URBMT, but the melphalan dose significantly impacts the kinetics of chimerism. Chimerism status evaluation at day 14 may be instrumental in predicting relapse after URBMT with the FM regimen.

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.

Trisomy 8 (+8) is one of the most common cytogenetic abnormalities in adult patients with acute myeloid leukemia (AML). However, the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in adult patients with AML harboring +8 remains unclear. To evaluate, the outcome and prognostic factors in patients with AML harboring +8 as the only chromosomal abnormality or in association with other abnormalities, we retrospectively analyzed the Japanese registration data of 631 adult patients with AML harboring +8 treated with allogeneic HSCT between 1990 and 2013. In total, 388 (61%) patients were not in remission at the time of HSCT. With a median follow-up of 38.5 months, the probability of overall survival and the cumulative incidence of relapse at 3 years were 40 and 34%, respectively. In the multivariate analysis, two or more additional cytogenetic abnormalities and not being in remission at the time of HSCT were significantly associated with a higher overall mortality and relapse. Nevertheless, no significant impact on the outcome was observed in cases with one cytogenetic abnormality in addition to +8. Although more than 60% of the patients received HSCT when not in remission, allogeneic HSCT offered a curative option for adult patients with AML harboring +8.

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.

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.

目的：母親から子どもへの「愛着」「養育」の構成因子を抽出し，項目の信頼性と妥当性を検討することである．方法：構成因子として「適応」「敏感性」「親密性」をあげ，それぞれに愛着的因子・養育的因子の定義づけを行い計6因子とした．文献および自己で作成した244項目を，定義に則って母性・助産の専門家と検討し60項目まで絞り込みを行った．1か月児，1歳6か月児を持つ母親344名への自記式質問紙による調査の結果から，項目の信頼性と妥当性の検討を行い，項目を絞り込んだ．結果：項目分析の結果を照らし合わせ，項目を入れ替えながらクロンバックのα係数を比較し，30項目を選出した．30項目のクロンバックのα係数は0.882であり，探索的因子分析では6因子が抽出された．結論：抽出された6因子は想定したものと完全に一致はしなかったが，愛着的因子・養育的因子には想定通り分かれており，項目の信頼性と妥当性は支持された．

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.

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.

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.

The effect of pressure on viability and the synthesis of bacterial cellulose (BC) by Gluconacetobacter xylinus ATCC53582 were investigated. G. xylinus was statically cultivated in a pressurized vessel under 0.1, 30, 60, and 100 MPa at 25 degrees C for 6 days. G. xylinus cells remained viable and retained cellulose producing ability under all the conditions tested, though the production of cellulose decreased with increasing the pressure. The BCs produced at each pressure condition were analyzed by field emission scanning electron microscopy (FE-SEM) and Fourier Transform Infrared (FT-IR). FE-SEM revealed that the widths of BC fibers produced under high pressure decreased as compared with those produced under the atmospheric pressure. By FT-IR, all the BCs were found to be of Cellulose type I, as the same as typical native cellulose. Our findings evidently showed that G. xylinus possessed a piezotolerant (barotolerant) feature adapting to 100 MPa without losing its BC producing ability. This was the first attempt in synthesizing BC with G. xylinus under elevated pressure of 100 MPa, which corresponded to the deep sea at 10,000 m.

We investigated the quantities and phylogenies of amoA genes of ammonia-oxidizing archaea (AOA) belonging to Crenarchaeota and ammonia-oxidizing bacteria (AOB) belonging to Betaproteobacteria in water columns and deep-ocean cold seep sediment in the northeastern Japan Sea with a competitive PCR (cPCR) assay. Water samples were collected at depths of 2000 m and 2956 m. Sediment samples were collected where white bacterial mats had developed. The cPCR analysis revealed five to ten times more AOA than betaproteobacterial AOB in both the water columns and sediment. The abundance of the crenarchaeotal amoA gene was estimated at 6×101 and 3×102 copies ml-1 in the water columns at depths of 2000 m and 2956 m, and 1×108 and 1×107 copies g-1 in pelagic brown sediment and black sediment, respectively. Most archaeal amoA clones from water column at 2000 m fell into the Deep Marine Group. Most archaeal amoA clones from pelagic brown sediment were less closely related to known environmental clones. Moreover, incubation experiments revealed nitrite production at 4°C and 10°C. The results indicate that psychrophilic AOA and AOB may be responsible for nitrification in the deep-ocean region of the northeastern Japan Sea.

3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier ((13)C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.

Deep-sea sediment samples were collected at a depth of 3,064 m in the Japan Sea. Microorganisms in the sediment sample were cultivated under several pressure conditions, and the high-pressure adapted microbes were isolated. Two of the isolates exhibited piezophilic growth profiles. This is the first report to show the presence of piezophiles in the Japan Sea.

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was sigma(70) containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the beta and beta' subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni(2+) affinity column. In vitro reconstitution using all proteins for the holoenzyme (alpha, beta, beta', sigma(70)) was carried out and the activity of the recombinant RNA polymerase was detected.

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.

Two strains of obligately piezophilic bacteria were isolated from sediment collected from the deepest cold-seep environment with chemosynthesis-based animal communities within the Japan Trench, at a depth of 7434 m. The isolated strains, JT7301 and JT7304 T , were closely affiliated with members of the genus Psychromonas on the basis of 16S rDNA sequence analysis. Hybridization values for DNA-DNA relatedness between these strains and the Psychromonas antarctica reference strain were significantly lower than that accepted as the phylogenetic definition of a species. The optimal temperature and pressure for growth of the isolates were 10 °C and 50 MPa and they produced both eicosapentaenoic acid (C20:5ω3) and docosahexaenoic acid (C22:6) in the membrane layer. Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Psychromonas species. The name Psychromonas kaikoae sp. nov. (type strain JT7304 T = JCM 11054 T = ATCC BAA-363 T ) is proposed. This is the first proposed obligately piezophilic species of the genus Psychromonas.

The dcw cluster of genes involved in cell division and cell wall synthesis from the piezophilic deep-sea bacterium Shewanella violacea was isolated and characterized. It comprises 15 open reading frames, of which the organization is mraZ-mraW-ftsL-ftsI-murE-murF-mraY-murD-ftsW-murG-murC-ftsQ-ftsA-ftsZ-envA, in that order. To analyze transcription upstream from the ftsZ gene, Northern blot and primer extension analyses were performed. The results showed that gene expression is not pressure dependent. Western blot analysis showed that the FtsZ protein is equally expressed under several pressure conditions in the range of atmospheric (0.1 MPa) to high (50 MPa) pressures. Using immunofluorescence microscopy, the FtsZ ring was observed in the center of cells at pressure conditions of 0.1 to 50 MPa. These results imply that the FtsZ protein function is not affected by elevated pressure in this piezophilic bacterium.

Cytochrome c₅ of pressure-sensitive Shewanella livingstonensis (SL cytc₅) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc₅ proteins are appropriate materials for understanding the protein stability mechanism.

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c 5 (SB cytc 5) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c 5 (SV cytc 5). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c 5 (SA cytc 5), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c 5 (SL cytc 5). The midpoint GdnHCl concentrations of SB cytc 5, SV cytc 5, SL cytc 5, and SA cytc 5 correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c 5 is related directly to its host's optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc 5 and SA cytc 5 are ancestors of the corresponding residues in piezophilic SB cytc 5 and SV cytc 5, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c 5 are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.

9 páginas.-- 3 figuras.-- referencias.-- El autor González Grau, Juan Miguel pertenece actualmente al Instituto de Recursos Naturales y Agrobiología de Sevilla, A new anaerobic, thermophilic, CO-utilizing marine bacterium, strain JMT, was isolated from a submarine hot vent in Okinawa Trough. Cells of strain JMT were non-motile thin straight rods, sometimes branching, with a cell wall of the Gram-positive type, surrounded with an S-layer. Chains of three to five cells were often observed. The isolate grew chemolithotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O-->CO2+H2) and organotrophically on peptone, yeast extract, starch, cellobiose, glucose, galactose, fructose and pyruvate, producing H2, acetate and CO2. Growth was observed from 50 to 80 degrees C with an optimum at 70 degrees C. The optimum pH was 6.8-7.1. The optimum concentration of sea salts in the medium was 20.5-25.5 g l(-1). The generation time under optimal conditions was 7.1 h. The DNA G+C content was 33 mol %. Growth of isolate JMT was not inhibited by penicillin, but ampicillin, streptomycin, kanamycin and neomycin completely inhibited growth. The results of 16S rDNA sequence analysis revealed that strain JMT belongs to the Thermoanaerobacter phylogenetic group within the Bacillus-Clostridium subphylum of Gram-positive bacteria but represents a separate branch of this group. On the basis of morphological and physiological features and phylogenetic data, this isolate should be assigned to a new genus, for which the name Carboxydobrachium is proposed. The type species is Carboxydobrachium pacificum; the type strain is JMT (= DSM 12653T)., The authors are grateful to G. A. Zavarzin and A. V. Lebedinsky, Institute of Microbiology, Russian Academy of Sciences, for helpful discussions. This work was supported by the Russian Foundation for Basic Research, grants nos 96-04-49463 and 99-04-48360, the ‘Biodiversity’ program of the Russian Ministry of Science and Technology, a NATO CLG grant to T. S. and F. T. R. and by NSF LEXEN Program support to F. T. R. and J. M. G.

3-isopropylmalate dehydrogenase (IPMDH)-encoding leuB genes were obtained from the obligate piezophile Shewanella benthica DB21MT-2 and non-piezophile Shewanella oneidensis MR-1. The genes were expressed in Escherichia coli and the proteins were purified using His-tag. The estimated kinetic parameters of these enzymes indicated that IPMDH of S. benthica DB21MT-2 is more tolerant of high pressure than that of S. oneidensis MR-1. Thus such an adaptation is one of the mechanisms bacteria utilize for survival at high pressures.

Two genes for alternative sigma factors, sigma(E2) and sigma(E3), classified in the extracytoplasmic function sigma family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli sigma(E) in both proteins. Core-binding analysis suggested that both proteins function as sigma factors.

Deep-sea soft sediments from trench systems and depths in the northwestern Pacific Ocean ranging from less than 300 to 10,897 m in depth have been analyzed for three target genera of actinomycetes: Micromonospora, Rhodococcus, and Streptomyces. Only culturable strains, recovered at atmospheric pressure on selective isolation media, have been examined to date. Maximum recoveries of culturable bacteria were greater that 10(7)/ml wet g sediment, but actinomycetes comprised a small proportion of this population (usually less than 1%). The target actinomycetes were isolated at all depths except from the Mariana Trench sediments. Actinomycete colonies were defined initially on the basis of colony morphologies, and preliminary identification then was made by chemotaxonomic tests. Pyrolysis mass spectrometry (PyMS) of deep-sea mycolic acid-containing actinomycetes gave excellent correspondence with numerical (phenetic) taxonomic analyses and subsequently was adopted as a rapid procedure for assessing taxonomic diversity. PyMS analysis enabled several clusters of deep-sea rhodococci to be distinguished that are quite distinct from all type strains. 16S rRNA gene sequence analysis has revealed that several of these marine rhodococci have sequences that are very similar to certain terrestrial species of Rhodococcus and to Dietzia. There is evidence for the intrusion of terrestrial runoff into these deep trench systems, and the inconsistency of the phenotypic and molecular taxonomies may reflect recent speciatiion events in actinomycetes under the high-pressure conditions of the deep sea. The results of DNA-DNA pairing experiments point to the novelty of Rhodococcus strains recovered from hadal depths in the Izu Bonin Trench. Biotransformation studies of deep-sea bacteria have focused on nitrile compounds. Nitrile-metabolizing bacteria, closely related to rhodococci, have been isolated that grow well at low temperature, high salt concentrations, and high pressures, suggesting that they are of marine origin or have adapted to the deep-sea environment.

We investigated for the first time the respiratory chain system of a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F. A membrane-bound ccb-type quinol oxidase, from cells grown at 60 MPa pressure, was purified to an electrophoretically homogeneous state. The purified enzyme complex consisted of four kinds of subunits with molecular masses of 98, 66, 18.5, and 15kDa, and it contained 0.96 mol of protoheme and 1.95mol of covalently bound heme c per mol of enzyme. Only protoheme in the enzyme reacted with CO and CN-, and the catalytic activity of the enzyme was 50% inhibited by 4 microM CN-. The isoelectric point of the native enzyme complex was determined to be 5.0. This enzyme was specifically induced only under conditions of elevated hydrostatic pressure, and high levels were expressed in cells grown at 60 MPa. The membranes isolated from cells grown at atmospheric pressure (0.1 MPa) exhibited high levels of both cytochrome c oxidase and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPDH2)-oxidase activity. These results suggest the presence of two kinds of respiratory chains regulated in response to pressure in the deep-sea bacterium DB-172F.

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.

The method of electrophoretic mobility shift assay under high-pressure conditions was improved using a high-pressure electrophoresis apparatus with capillary narrow-tube gel. It was found that the protein-DNA complex in the gel was stained as a high-resolution spot with ethidium bromide. Using this method, it was found that the behavior under high-pressure conditions of the protein-DNA complex composed of NtrC protein and its target promoter DNA is important for the pressure-regulated transcription process, and it was confirmed that the complex was dissociated above a pressure of 70 MPa.

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.

Hydrostatic pressure analysis is an ideal approach for studying protein dynamics and hydration. The development of full ocean depth submersibles and high pressure biological techniques allows us to investigate enzymes from deep-sea organisms at the molecular level. The aim of this review was to overview the thermodynamic and functional characteristics of deep-sea enzymes as revealed by pressure axis analysis after giving a brief introduction to the thermodynamic principles underlying the effects of pressure on the structural stability and function of enzymes.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

The facultative piezophile Shewanella violacea DSS12 is known to alter its respiratory components under the influence of hydrostatic pressure during growth, suggesting that it has a respiratory system that functions in adaptation to high pressure. We investigated the pressure- and temperature-dependencies of the respiratory terminal oxidase activity of the membrane of S. violacea relative to non-piezophilic Shewanella species. We observed that the activity in the membrane of S. violacea was more resistant to high pressure than those of non-piezophilic Shewanella even though DSS12 was cultured under atmospheric pressure. On the other hand, the temperature dependency of this activity was almost the same for all of the tested strain regardless of optimal growth temperature. Both high pressure and low temperature are expected to lower protein flexibility, causing a decrease in enzyme activity, but the results of this study suggest that the mechanism maintaining enzyme activity under high hydrostatic pressure is different from that at low temperature. Additionally, the responses of the activity to the pressure- and temperature-changes were independent of membrane lipid composition. Therefore, the piezotolerance of the respiratory terminal oxidases of S. violacea is perhaps dependent on the properties of the protein itself and not on the lipid composition of the membrane. Our observations suggest that S. violacea constitutively express piezotolerant respiratory terminal oxidases that serve adaptation to the deep-sea environment.

The deep-sea denitrifier Pseudomonas sp. strain MT-1 has two distinct gene clusters encoding dissimilatory nitrate reductases, periplasmic nitrate reductase (Nap) and membrane-bound nitrate reductase (Nar). In order to investigate the physiological roles of these enzymes, we determined the nitrate reductase activity of the soluble and membrane fractions from MT-1 and the type strain of Pseudomonas stutzeri (closely related with MT-1) grown under various conditions. In MT-1, the activities of both fractions were highest when the cells were grown anaerobically in the presence of nitrate under atmospheric pressure. However, the activity of the soluble fraction decreased when the cells were grown under high pressure, whereas that of membrane fraction remained constant. Further, the activity of the soluble fraction decreased when the enzyme reaction was performed at low temperature, although that of membrane fraction was not similarly affected. Additionally, the results of RT-PCR showed that expression of the nar genes was strongly induced under high pressure. In contrast, P. stutzeri(T) showed no such response following a shift in growth pressure. These results suggest that MT-1 possesses a special mechanism for adaptation to the low-temperature and high-pressure environments of the deep sea, and that Nar is the main dissimilatory nitrate reductase in MT-1 in such environments.

We obtained DNA fragments encoding putative aminotransferases possibly involved in the biosynthesis of aminoglycoside antibiotics from deep-sea sediments of the northwest Pacific Ocean by nested PCR, and 34 individual genes (total 89 clones) were identified. About half of the deep-sea sequences showed similarity with genes of known aminoglycoside-producers, but others were deep-sea specific genes. Furthermore, we found that temperature-gradient gel electrophoresis (TGGE) can be an effective tool in the analysis of these DNA fragments.

To understand reductive genome evolution (RGE), we comparatively analyzed the recently reported small genomes of two chemoautotrophic, intracellular symbionts of deep-sea clams, Calyptogena okutanii and C. magnifica. Both genomes lack most genes for DNA recombination and repair such as recA and mutY. Their genome architectures were highly conserved except one inversion. Many deletions from small (

The nap gene cluster encoding periplasmic nitrate reductase was identified from Pseudomonas sp. strain MT-1, a deep-sea denitrifier isolated from the Mariana Trench. The ORFs identified were highly homologous with those of Pseudomonas stutzeri, but the cluster included only four ORFs (napDABC), less than those in other organisms. For other bacteria, some additional small ORFs (such as napE, napF, napG, napH, and napK) are found in the nap gene cluster, although their physiological function is still unclear. The soluble fraction of MT-1 grown under denitrifying condition showed significant nitrate reductase activity. This observation suggests that the periplasmic nitrate reductase encoded by the gene cluster identified in this study is functional. The activity was highest when the organism was grown under denitrifying conditions, suggesting that the enzyme participates in dissimilatory nitrite reduction.

An obligately piezophilic bacterium was isolated from sediment collected from the bottom of the Japan Trench at a depth of 7542 m. The isolated strain, designated K41GT, was closely affiliated with members of the genus Psychromonas on the basis of 16S rRNA gene sequence analysis. Levels of DNA–DNA relatedness between strain K41GT and Psychromonas reference strains were significantly lower than that accepted as the phylogenetic definition of a species. The optimal temperature and pressure for growth of strain K41GT were 6 °C and 60 MPa, respectively. The DNA G+C content was 39.1 mol%. Whole-cell fatty acids consisted of significant amounts of unsaturated fatty acids C16 : 1 (37 %) and C14 : 1 (17 %), saturated fatty acid C16 : 0 (31 %) and polyunsaturated fatty acid C22 : 6 (8 %). Based on the taxonomic differences observed, strain K41GT is considered to represent a novel obligately piezophilic Psychromonas species. The name Psychromonas hadalis (type strain, K41GT=JCM 11830T=ATCC BAA-638T) is proposed. This is the second species of obligately piezophilic bacteria to be proposed in the genus Psychromonas.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between −0.2 and −0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications., Yeast Saccharomyces cerevisiae were selectively attached on the negative potential-applied indium tin oxide/glass electrode. Mechanisms of the yeast cell attachment involve Flocculin Flo10 proteins., Graphical Abstract Figure. Yeast Saccharomyces cerevisiae were selectively attached on the negative potential-applied indium tin oxide/glass electrode. Mechanisms of the yeast cell attachment involve Flocculin Flo10 proteins.

Chronic myelogenous leukemia (CML) is a hematological malignancy that is characterized by the chromosome anomaly, t(9;22)(q34;q11). By this chromosomal translocation, a novel activated tyrosine kinase, BCR-ABL chimeric protein, is generated, and the protein is causative of the disease. Recently, Imatinib mesylate targeting to a BCR-ABL chimeric protein has been developed, and shown to achieve complete remission at a high rate. Patients are currently required to receive a fixed dose, 400 mg daily; however, it is possible that some of patients can maintain their remission with reduced doses of imatinib. In this study, we determined levels of BCR-ABL transcript in CML patients by real-time quantitative polymerase chain reaction analysis, and explored the possibility of individualization of therapeutic doses of imatinib. Thirty-five CML patients, including 17 newly diagnosed patients, 16 patients pre-treated with interferon-alpha, and 2 relapsed patients after allogeneic transplantation, were treated with imatinib. Complete cytogenetic response was achieved in 31 (89%) patients. Major molecular response (MMR) was achieved in 21 (60%). Complete molecular response (CMR) was achieved in 7 (20%). Imatinib was discontinued in 2 patients, one patient with MMR due to noncompliance and other patient sustaining CMR, but both patients relapsed 7 and 13 months later, respectively. The doses of imatinib were reduced in 7 patients due to its side effects, but 4 out of the 7 patients have sustained MMR, and 2 of them have sustained CMR for more than 23 months. These results indicate that some patients are able to maintain MMR with low-dose imatinib.

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0–120 g l−1, with the optimum at 10–20 g l−1. The temperature range for growth at pH 7·0 was 4–50 °C, with the optimum at 37–40 °C. The optimum pH for growth was 7·0–8·0. The optimum pressure for growth was 0·1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2T (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA–DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

The genes of cytochrome bd-encoding cydAB were identified from a deep-sea bacterium Shewanella violacea DSS12. These showed significant homologies with known cydAB gene sequences from various organisms. Additionally, highly conserved regions that are important for the enzymatic function were also conserved in cydA of S. violacea. Based on the results, transcriptional analysis of cydAB operon and cydDC operon (required for assembly of cytochrome bd) of S. violacea in microaerobic condition was performed under the growth condition of various pressures. The gene of cydA was expressed even under the condition of atmospheric pressure and its expression was enhanced with pressurization. On the other hand, the expression of cydC was strongly depressed under the condition of atmospheric pressure compared with the case under high pressure. It appeared spectrophotometrically that loss of cytochrome bd in S. violacea under atmospheric pressure shown in previous study is caused mainly by the loss of cydDC. Further, under the growth condition of atmospheric pressure, either less amount or no d-type cytochrome was expressed compared with the case of high-pressure condition even if the organism was grown under alkaline condition or in the presence of uncoupler, which are the inducible condition of d-type cytochrome in Escherichia coli. These results suggested that the significant amount of d-type cytochrome expression is specific event under the growth condition of high pressure.

A novel piezotolerant, mesophilic, facultatively anaerobic, organotrophic, polarly flagellated bacterium (strain LT13aT) was isolated from a deep sediment layer in the Nankai Trough (Leg 190, Ocean Drilling Program) off the coast of Japan. This organism used a wide range of organic substrates as sole carbon and energy sources: pyruvate, glutamate, succinate, fumarate, lactate, citrate, peptone and tryptone. Oxygen, nitrate, fumarate, ferric iron and cystine were used as electron acceptors. Maximal growth rates were observed at a hydrostatic pressure of 10 MPa. Hydrostatic pressure for growth was in the range 0·1–50 MPa. Predominant cellular fatty acids were 16 : 1ω7c, 15 : 0 iso, 16 : 0 and 13 : 0 iso. The G+C content of the DNA was 44·9 mol%. On the basis of 16S rRNA gene sequences, strain LT13aT was shown to belong to the γ-Proteobacteria, being closely related to Shewanella putrefaciens (98 %), Shewanella oneidensis (97 %) and Shewanella baltica (96 %). Levels of DNA homology between strain LT13aT and S. putrefaciens, S. oneidensis and S. baltica were T represents a novel species. Genetic evidence and phenotypic characteristics showed that isolate LT13aT constitutes a novel species of the genus Shewanella. Because of the deep origin of the strain, the name Shewanella profunda sp. nov. is proposed, with LT13aT (=DSM 15900T=JCM 12080T) as the type strain.