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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 previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ± 0.1 MPa and a volume change of −225 ± 24 mL mol−1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and −30 mL mol−1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Earlier reports suggested that umbilical cord blood transplantation (UCBT) for aplastic anemia (AA) was feasible in alternative transplantation. To identify differences in outcomes of UCBT and HLA-matched or mismatched unrelated bone marrow transplantation (UBMT) in adults with AA, we analyzed registry data of the Japan Society for Hematopoietic Cell Transplantation and compared results of UCBT (n = 69) to 8/8-matched (n = 101), 7/8-matched (n = 65), or 6/8-matched (n = 37) UBMT. The transplantation period was from 2002 to 2012, and patients 16 years or older with AA were eligible. Median ages were 49, 35, 28, and 30 years for UCBT, 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively. In multivariate analysis, risk of mortality was lower for 8/8-matched UBMT compared with that of UCBT (hazard ratio [HR], .55; 95% confidence interval [CI], .32 to .94; P = .029), adjusted for age and graft-versus-host disease (GVHD) prophylaxis, which were other associated factors. Mortality risks of 7/8-matched UBMT (HR, .55; 95% CI, .29 to 1.02) or 6/8-matched UBMT (HR, .67; 95% CI, .32 to 1.39) were not significantly different from those of UCBT. Risks of grade 3 or 4 acute and chronic GVHD were not different among the 4 groups. The most prevalent cause of death was graft failure in UCBT and 6/8-matched UBMT and infection in 8/8-matched and 7/8-matched UBMT. Under 40 years old,survival of UCBT was similar to that of UBMT (76%, 79%, 83%, and 83% for UCBT and 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively, at 3 years), adjusted for transplantation period, which was another associated factor; however, for ages over 40 years, that of UCBT tended to be lower (47%, 64%, 64%, and 75% for UCBT, 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively, at 3 years). To conclude, these data suggest that UCBT could be an alternative treatment option for younger adults when matched sibling or adequate UBMT donors are not available.

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.

Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation (allo-HSCT). Recently, it was reported that CMV reactivation is associated with a decreased risk of relapse in patients with acute myeloid leukemia (AML). The aim of this study was to evaluate the impact of early CMV reactivation on the incidence of disease relapse after allo-HSCT in a large cohort of patients. The Japan Society for Hematopoietic Cell Transplantation's Transplantation-Related Complication Working Group retrospectively surveyed the database of the Transplant Registry Unified Management Program at the Japan Society for Hematopoietic Cell Transplantation. Patients with AML (n = 1836), acute lymphoblastic leukemia (ALL, n = 911), chronic myeloid leukemia (CML, n = 223), and myelodysplastic syndrome (MDS, n = 569) who underwent their first allo-HSCT from HLA-matched related or unrelated donors between 2000 and 2009 and who survived without disease relapse until day 100 after transplantation were analyzed. Patients who received umbilical cord blood transplantation were not included. Patients underwent surveillance by pp65 antigenemia from the time of engraftment, and the beginning of preemptive therapy was defined as CMV reactivation. Cox proportional hazards models were used to evaluate the risk factors of relapse, nonrelapse, and overall mortality. CMV reactivation and acute/chronic graft-versus-host disease (GVHD) were evaluated as time-dependent covariates. CMV reactivation was associated with a decreased incidence of relapse in patients with AML (20.3% versus 26.4%, P = .027), but not in patients with ALL, CML, or MDS. Among 1836 patients with AML, CMV reactivation occurred in 795 patients (43.3%) at a median of 42 days, and 436 patients (23.7%) relapsed at a median of 221 days after allo-HSCT. Acute GVHD grades II to IV developed in 630 patients (34.3%). By multivariate analysis considering competing risk factors, 3 factors were significantly associated with a decreased risk of AML relapse and 1 factor with an increased risk of AML relapse: CMV reactivation (hazard ratio [HR], .77; 95% confidence interval [CI], .59 to .99), unrelated donor compared with related donor (HR, .59; 95% CI, .42 to .84), development of chronic GVHD (HR, .77; 95% CI, .60 to .99), and pretransplantation advanced disease status compared with standard disease status (HR, 1.99; 95% CI, 1.56 to 2.52). However, CMV reactivation was associated with increased nonrelapse mortality (HR, 1.60; 95% CI, 1.18 to 2.17) and overall mortality (HR, 1.37; 95% CI, 1.11 to 1.69). A beneficial effect of CMV reactivation on subsequent risk of relapse was observed in patients with AML but not in those with other hematological malignancies. However, this benefit was nullified by the increased nonrelapse mortality. The underlying mechanism is unclear; however, immunological activation against CMV reactivation plays an essential role in this association. Thus, immune augmentation treatment options, including vaccination and adoptive T cell transfer, may be useful to take advantage of the efficacy of CMV reactivation with minimal increase in nonrelapse mortality.

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.

Cracking growth tests were conducted in high-temperature water containing hydrogen peroxide (H2O2) at 561–423 K to evaluate the effects of H2O2 on stress corrosion cracking (SCC) of stainless steel (SS) at temperature lower than the boiling water reactor (BWR) operating temperature. Small compact tension (CT) specimens were prepared from thermally sensitized type 304 SS. Despite the observation of only a small portion intergranular SCC (IGSCC) near the side groove of the CT specimen at 561 K in high-temperature water containing 100 ppb H2O2, the IGSCC area expanded to the central region of the CT specimens at 423 and 453 K. Effects of H2O2 on SCC appeared intensely at temperature lower than the BWR operating temperature because of a reduction in the thermal decomposition of H2O2. To estimate the environment in the cracks, outer oxide distribution on the fracture surface and the fatigue pre-crack were examined by laser Raman spectroscopy and thermal equilibrium calculation was performed.

Crack growth tests were performed in high-temperature water containing hydrogen peroxide (H 2 O 2 ) to evaluate the relationships between the crevice structure and H 2 O 2 on stress corrosion cracking (SCC) growth morphology of stainless steel (SS). Small compact tension (CT) specimens were prepared from thermally sensitized type 304 SS. 20–300 ppb H 2 O 2 was injected into the high-temperature water at 561 K. Intergranular SCC (IGSCC) and transgranular SCC were observed near the side grooves and the central region of the original CT specimens, respectively. Chevron notches were removed from the CT specimens after fatigue pre-crack introduction. Owing to pre-crack shortening, the IGSCC area expanded to the central region of the CT specimens and increased with H 2 O 2 concentration. The effects of H 2 O 2 on SCC appeared intensely near the surfaces exposed to high levels of H 2 O 2 . Microanalysis and distribution examination of oxide layers were performed and the percentage of H 2 O 2 remaining in the crack was calculated.

Development of a high performance proton beam window material is one of the critical issues for the deployment of the accelerator-driven transmutation system (ADS) with liquid Pb–Bi eutectic as a spallation target and coolant. In the present study, we applied 20% cold work treatment to JPCA austenitic stainless steel and investigated it from the corrosion behavior viewpoint. The corrosion test of 20% cold-worked JPCA SS has been carried in the JLBL-1 (JAEA Lead–Bismuth Loop-1) apparatus. The maximum temperature, the temperature difference, the flow velocity and the exposure time of the liquid Pb–Bi were 450 °C, 100 °C, 1 m/s, and 1000 h, respectively. For comparison analysis, JPCA SS without cold working was also tested in the same time and conditions with the 20% cold-worked JPCA SS. The results showed a different corrosion behavior between the JPCA SS without and with cold working. As for the JPCA SS without cold working, Pb–Bi penetrated into the matrix through a ferrite layer which was formed because of constituent metals dissolution from the matrix into Pb–Bi. As for the 20% cold-worked JPCA SS, dissolution attack occurred only partially and formed localized superficial pitting corrosion. It was found that the different corrosion behavior occurred because the cold working induced a structure transformation from γ-austenite to α′-martensite and affected the corrosion resistance of the JPCA SS in flowing Pb–Bi at 450 °C.

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 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.

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.

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.

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.

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.

The biochemistry of piezophilic bacteria is unique in that piezophiles produce polyunsaturated fatty acids (PUFAs). A pertinent question is if piezophilic bacteria synthesize PUFA de novo, through dietary uptake, or both. This study was undertaken to examine the biosynthesis and cellular uptake of PUFAs by piezophilic bacteria. A moderately piezophilic (Shewanella violacea DSS12) and two hyperpiezophilic bacteria (S. benthica DB21MT-2 and Moritella yayanosii DB21MT-5) were grown under 50 MPa (megapascal) and 100 MPa, respectively, in media containing marine broth 2216 supplemented with arachidonic acid (AA, sodium salt) and/or antibiotic cerulenin. There was active uptake and cellular incorporation of AA in the hyperpiezophilic bacteria DB21MT-2 (14.7% of total fatty acids) and DB21MT-5 (1.4%), but no uptake was observed in DSS12. When cells were treated with cerulenin, all three strains incorporated AA into cell membranes (13-19%). The biosynthesis of monounsaturated fatty acids was significantly inhibited (10-37%) by the addition of cerulenin, whereas the concentrations of PUFAs increased by 2-4 times. These results suggest that piezophilic bacteria biosynthesize and/or incorporate dietary polyunsaturated fatty acids that are important for their growth and piezoadaptation. The significance of these findings is also discussed in the context of phenotypic classification of piezophiles.

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-delta and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-delta, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.

Two extremely piezophilic bacteria, DB21MT-2 and DB21MT-5, isolated from sediments of the Mariana Trench at 11,000 m, were grown in the laboratory under low-temperature (10 jC) and high-pressure (70 MPa) conditions. Phospholipid esterlinked fatty acids (PLFA) were analyzed by gas chromatography/mass spectrometry (GC/MS) and isotopic compositions of individual fatty acids were determined by gas chromatography isotope ratio mass spectrometry (GC-IRMS) in order to determine isotopic fractionation effects during biosynthesis. The piezophiles are characterized by fatty acids with carbon chains ranging from 14 to 22 carbons in length and by the presence of abundant polyunsaturated fatty acids (PUFA). Fatty acids from each culture exhibit a wide range of isotopic compositions (d 13 C vs. Pee Dee Belemnite (PDB)). With two exceptions (C15:0 and C17:1 from DB21MT-2), all fatty acids are depleted in 13 C relative to that of bulk carbon in the bacterial growth substrate. In addition, strain DB21MT-2 shows systematically higher (less negative) d 13 C values than strain DB21MT-5. The implications for isotope fractionation in fatty acid biosynthesis and the interpretation of isotopic composition of sedimentary fatty acids are discussed. It is suggested that the same type of microorganisms could have rather different d 13 C under the same growth conditions, and that sedimentary fatty acids with distinct d 13 C values do not necessarily have to originate from different organisms. Interpretation of d 13 C values of sedimentary biomarkers may be complicated by such large variations in d 13 Co f

We investigated whether vitamin E (VE) supplementation alleviates collagen-induced arthritis in its stages by altering immune functions in DBA/1J mice. Mice were divided into 4 groups; control (VE 30 IU/kg in diet), A-VE0, A-VE30 and A-VE500 (animals with induced arthritis given VE 0, VE 30, VE 500 IU/kg in diet respectively). Arthritis was induced with Type II Collagen (TIIC). The extent of arthritis in the joint did not decline in neither A-VE0 nor A-VE500 groups compared with A-VE30. The proliferation of splenocytes stimulated with ConA was suppressed in both A-VE0 and A-VE500 groups. The production of Th1 cytokine stimulated with TIIC was increased in both A-VE0 and A-VE500 groups, but production of Th2 cytokine was not changed. Our observations suggest that VE supplementation does not always improve the progress of arthritis. The influence of VE on cytokine balance in autoimmune (Th1 predominant) arthritis should be further investigated.

Five passenger vehicles used around the Detroit area were submitted for corrosion investigation. They were manufactured in 1989 and 1991. Panels forming the body shell were classified into five categories according to the corrosion environment, and their cosmetic corrosion and perforation corrosion were evaluated. It was confirmed that exterior panels made of two-sided zinc coated steel sheet has excellent performance in cosmetic corrosion, and it was shown that the corrosion depth at lapped parts under low humid conditions decreases with increased zinc coating weight. For precoated panels with zinc coating more than 45 g/m 2 , the degree of corrosion was very low.

Although decreased T-cell function has been observed in obese human subjects and genetically obese animals, the precise role of immune functions in obesity is still unclear. To investigate immune functions in obesity, we examined the proliferative responses of splenic lymphocytes and their capacity to produce cytokines in the presence or absence of leptin, the protein produced by the obese gene, in diet-induced obese and control mice. For induction of obesity, C57BL/6J mice were fed a high-fat diet for 13 weeks. In mice fed the high-fat diet, body weight, fat pad weight, and tumor necrosis factor (TNF) alpha production by adipocytes were significantly increased relative to mice fed the normal diet. Lipopolysaccharide (LPS) stimulated proliferation of cultured splenocytes from diet-induced obese mice was also increased. However, production of interleukin (IL)-2 by splenic lymphocytes from obese mice was suppressed, whereas interferon (IFN)-gamma and IL-4 production was increased. Exogenous lepitn regulated the cytokine production by cultured splenocytes from control and obese mice, respectively (upregulation of IFN-gamma and downregulation of IL-2 in control mice, and downregulation of IL-4 in obese mice). These results suggest that changes in cytokine production by splenic lymphocytes in obesity are indicative of altered immune functions that might contribute to related complications, although the effect of difference in nutrient intake (macro and micro) may also have contributed to the changes.

The membrane phospholipids of two barophilic bacteria, DB21MT-2 and DB21MT-5, isolated from sediments from the Marianas Trench at 11,000 m were structurally characterized and quantitatively determined by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS). Phospholipids detected in the barophiles were distributed in five different classes: phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), diphosphatidylglycerol (DPG), and phosphatidylethanolamine (PE) and its methylated forms phosphatidylmethylethanolamine (PME) and phosphatidyldimethylethanolamine (PDME). Concentrations of phospholipids/phospholipid groups range from 5.3 to 4.583 μg/g (dry weight). A unique aspect of the phospholipid profiles of the barophilic bacteria was the wide distribution of polyunsaturated fatty acids 20 : 5 (in DB21MT-2) and 22 : 6 (in both DB21MT-5 and DB1MT-2) on the sn -1 and mostly on the sn -2 position of the phospholipids. The results suggest that the adaptation of the barophiles to low temperature and high hydrostatic pressure influenced the synthesis of phospholipids containing polyunsaturated fatty acids. It was also observed that the polyunsaturated fatty acids were associated with almost every phosphatidylglycerol (PG) molecule. Presumably, the larger head group of PG would be expected to cause greater disruption in acyl chain packing within the membrane bilayer and thereby lower the transition temperature in response to the additive effects of low temperature and high pressure. The detection of phospholipids with polyunsaturated fatty acids also has important geochemical implications for paleoenvironment reconstruction and for determining the surface water biological productivity of the ocean. It seems that psychrophilic and barophilic bacteria may be major sources of polyunsaturated fatty acids to the deep-sea sediments, given the fact that the vertical flux of polyunsaturated fatty acids from surface water plankton decreased rapidly with depth.

We have cloned the gene encoding RNA polymerase alpha subunit from a gene library of deep-sea barophilic bacterium strain DB6705. The clone contains the genes for ribosomal protein S4, RNA polymerase subunit alpha and ribosomal protein L17 in this order. The alpha gene has 328 amino acids and a molecular mass of 36 100 Da with 86.9% identity to Escherichia coli alpha gene. Differences between the two sequences were mainly in the N-terminal portion of the alpha subunit, which is involved in the assembly of the core RNA polymerase; while the 87 C-terminal residues, which form a region involved in contact with some positive regulators and rrnB P1 promoter region called UP-element, were identical in the both strain. Plasmid encoding the alpha subunit with an N-terminal hexahistidine tag was constructed. Using the plasmid, the recombinant fusion alpha subunit was overexpressed and successfully purified to near homogeneity.

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, pressure, however, is an exception. Development of the pressure sensitive fluorescent protein would blaze a new path to the future biology. Recently, we enhanced the pressure sensitivity of yellow fluorescent protein (YFP) by inserting several glycines into it. Our mutation enhanced the interaction between the chromophore and the solvent that is responsible for pressure changing. In response to changes in pressure, a spectrum shift and an intensity change of the fluorescence was observed, indicating applicability of the present YFP for measuring the intracellular pressure on a live specimen. By measuring the fluorescence of the mutant YFP, we succeeded in measuring the intracellular pressure changes that follow actin filament disruption in living cells. This study shows that our mutation enhanced unknown properties of YFP that may allow us to measure the intracellular pressure.View Large Image | View Hi-Res Image | Download PowerPoint Slide