Abstract  We developed methods for evaluating the ntial inhibition of human cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2 C9, CYP2 C19, CYP2D6, CYP2E1 and CYP3A4, using pooled human liver microsomes (HLMs). The CYP inhibition assay used substrate cocktail sets [set A: phenacetin for CYP1A2, coumarin for CYP2A6, (S)-(+)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4; set B: bupropion for CYP2B6, tolbutamide for CYP2C9, chlorzoxazone for CYP2E1, and testosterone for CYP3A4] with quantitation by liquid chromatography-tandem mass spectrometry. A direct inhibition assay was performed with the substrate cocktails without β-nicotinamide adenine dinucleotide phosphate (NADPH) pre-incubation, and a metabolism-dependent inhibition (MDI) assay was performed after 30 min of pre-incubation with NADPH in HLMs. MDI was identified based on the half-maximal inhibitory concentration (IC(50)) shifts. The IC(50) values of the direct inhibitors determined using the probe substrate cocktails were in good agreement with previously reported values. Eight metabolism-dependent inhibitors including furafylline, 8-methoxypsoralen, tienilic acid, ticlopidine, fluoxetine, paroxetine, disulfiram and verapamil against CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively, resulted in significant IC(50) shifts (≥2.5-fold) after pre-incubation. Thus, these CYP inhibition assays are considered to be useful tools for evaluating both direct inhibition and MDI at an early stage of the drug discovery and development process.

Abstract  Suspended particles in a system made up of Aldrich humic acids (HAs) in water account for about 13% of the total HA mass, 10-11% of the organic carbon and 9-11% of radiation extinction in the UVA region. Extinction would be made up of radiation scattering (less than one third) and absorption (over two thirds). The contribution of particles to the degradation rates of trimethylphenol and furfuryl alcohol (FFA) (probes of triplet states and (1)O(2), respectively) was lower than 10% and possibly negligible. The results indicate that triplet states and (1)O(2) occurring in the solution bulk are mostly produced by the dissolved HA fraction. Experimental data would not exclude production of (1)O(2) in particle hydrophobic cores, unavailable for reaction with FFA. However, the limited to negligible particle fluorescence places an upper limit to particle core photoactivity.

Abstract  The aim of this study was to investigate the chemical composition and the odor-active components of volatile oils from three edible mushrooms, Pleurotus ostreatus, Pleurotus eryngii, and Pleurotus abalonus, which are well-known edible mushrooms. The volatile components in these oils were extracted by hydrodistillation and identified by GC/MS, GC-olfactometry (GC-O), and aroma extract dilution analysis (AEDA). The oils contained 40, 20, and 53 components, representing 83.4, 86.0, and 90.8% of the total oils in P. ostreatus, P. eryngii, and P. abalonus, respectively. Odor evaluation of the volatile oils from the three edible mushrooms was also carried out using GC-O, AEDA, and odor activity values, by which 13, eight, and ten aroma-active components were identified in P. ostreatus, P. eryngii, and P. abalonus, respectively. The most aroma-active compounds were C8 -aliphatic compounds (oct-1-en-3-ol, octan-3-one, and octanal) and/or C9 -aliphatic aldehydes (nonanal and (2E)-non-2-enal).

Abstract  BACKGROUND: Adverse effects of industrially produced trans fatty acids (iTFAs) on the risk of coronary artery disease are well documented in the scientific literature; however, effects of naturally occurring trans fatty acids (TFAs) from ruminant animals (rTFA), such as vaccenic acid (VA) and cis-9,trans-11 conjugated linoleic acid (c9,t11-CLA), are less clear. Although animal and cell studies suggest that VA and c9,t11-CLA may be hypocholesterolemic and antiatherogenic, epidemiologic data comparing rTFAs and iTFAs are inconsistent, and human intervention studies have been limited, underpowered, and not well controlled.

OBJECTIVE: We determined the effects of VA, c9,t11-CLA, and iTFA, in the context of highly controlled diets (24 d each), on lipoprotein risk factors compared with a control diet.

RESULTS: We conducted a double-blind, randomized, crossover feeding trial in 106 healthy adults [mean ± SD age: 47 ± 10.8 y; body mass index (in kg/m(2)): 28.5 ± 4.0; low-density lipoprotein (LDL) cholesterol: 3.24 ± 0.63 mmol/L]. Diets were designed to have stearic acid replaced with the following TFA isomers (percentage of energy): 0.1% mixed isomers of TFA (control), ∼3% VA, ∼3% iTFA, or 1% c9,t11-CLA. Total dietary fat (34% of energy) and other macronutrients were matched. Total cholesterol (TC), LDL cholesterol, triacylglycerol, lipoprotein(a), and apolipoprotein B were higher after VA than after iTFA; high-density lipoprotein (HDL) cholesterol and apolipoprotein AI also were higher after VA. Compared with control, VA and iTFA both increased TC, LDL cholesterol, ratio of TC to HDL cholesterol, and apolipoprotein B (2-6% change; P < 0.05); VA also increased HDL cholesterol, apolipoprotein AI, apolipoprotein B, and lipoprotein(a) (2-6% change; P < 0.05), whereas iTFA did not. c9,t11-CLA lowered triacylglycerol (P ≤ 0.01) and had no effect on other lipoprotein risk factors.

CONCLUSIONS: With respect to risk of cardiovascular disease, these results are consistent with current nutrition labeling guidelines, with the requirement of VA, but not c9,t11-CLA, to be listed under TFA on the Nutrition Facts Panel. This trial was registered at clinicaltrials.gov as NCT00942656.

Abstract  The objective of this research was to study the delta-9 desaturation of individual trans (t) fatty acids that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on lipogenic gene expression in adipocytes. It was hypothesized that delta-9 desaturation and lipogenic properties of t-18:1 isomers depend on the position of double bond. Differentiated 3T3-L1 adipocytes were treated with 200 µM of t6-18:1, t9-18:1, t11-18:1, t13-18:1 or t16-18:1, cis (c)-9 18:1 or bovine serum albumin (BSA) vehicle control for 48 h. Cells were then harvested for fatty acid and gene expression analyses using gas chromatography and quantitative PCR respectively. Among t-18:1 isomers, t13-18:1 and t11-8:1 had the greatest percent delta-9 desaturation (44 and 41 % respectively) followed by t16-18:1 and t6-18:1 (32 and 17 % respectively), while c9-18:1 and t9-18:1 did not undergo delta-9 desaturation. Trans9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including fatty acid synthase and stearoyl-CoA desaturase-1 (P < 0.05), whereas the expression of these genes were not affected with other t-18:1 isomers (P > 0.05). Consistent with gene expression results, t9-18:1 increased the de novo lipogenic index (16:0/18:2n-6) compared with control cells and increased delta-9 desaturation index (c9-16:1/18:0) compared to other t-18:1 isomers (P < 0.05). The current study provides further evidence that the predominant trans fatty acid in PHVO (t9-18:1) has isomer specific lipogenic properties.

Abstract  Histone deacetylases (HDAC) are metal-dependent enzymes and considered as important targets for cell functioning. Particularly, higher expression of class I HDACs is common in the onset of multiple malignancies which results in deregulation of many target genes involved in cell growth, differentiation and survival. Although substantial attempts have been made to control the irregular functioning of HDACs by employing various inhibitors with high sensitivity towards transformed cells, limited success has been achieved in epigenetic cancer therapy. Here in this study, we used ligand-based pharmacophore and 2-dimensional quantitative structure activity relationship (QSAR) modeling approaches for targeting class I HDAC isoforms. Pharmacophore models were generated by taking into account the known IC50 values and experimental energy scores with extensive validations. The QSAR model having an external R2 value of 0.93 was employed for virtual screening of compound libraries. 10 potential lead compounds (C1-C10) were short-listed having strong binding affinities for HDACs, out of which 2 compounds (C8 and C9) were able to interact with all members of class I HDACs. The potential binding modes of HDAC2 and HDAC8 to C8 were explored through molecular dynamics simulations. Overall, bioactivity and ligand efficiency (binding energy/non-hydrogen atoms) profiles suggested that proposed hits may be more effective inhibitors for cancer therapy.

Abstract  The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.

Abstract  The size, surface charge, and microstructure of lipoplexes comprising cationic lipids and nucleic acids are important factors for transfection efficiency. As these properties are largely determined by the cationic lipids used, a number of studies on the relationship between cationic lipids and the transfection efficiency have been reported. Among the many cationic lipids, lipids with multivalent cationic head groups are expected to be potent transfection reagents. Here, we prepared calix[4]arene-based lipids with different alkyl chain lengths from C3 to C15 and evaluated the relationship between the alkyl chain length and the transfection efficiency. C6 lipoplexes exhibited the highest transfection efficiency among all lipoplexes. The gene expression with C9 and C12 lipoplexes was slightly lower than that with C6 lipoplexes. C3 lipoplexes hardly induced gene expression, while C15 lipoplexes exhibited no complexation with plasmid DNA. Although all lipoplexes exhibited nearly identical characteristics, they exhibited different behaviours in terms of the interactions between the lipoplexes and anionic micelles comprising phosphatidylserine, a model of endosomal vehicle. After mixing with phosphatidylserine micelles, C6 lipoplexes released the bound plasmid DNA at pH 5 but not at pH 7, indicating that they can interact with the late endosomal membrane after being incorporated into cells. No plasmid DNA was released from C9 or C12 lipoplexes at either pH values. Thus, the alkyl chain length of cationic lipids is related to their interaction with the endosomal compartment and can provide a basis for the design of novel transfection reagents.

Abstract  There are difficulties in conducting worldwide medical device development simultaneously because each country and/or region has their own medical device regulations. However, to aid globalization of the medical device market, and to quickly provide innovative medical devices to patients, attempts have been made to encourage harmonization and convergence of medical device regulations. 'Harmonization by doing' is a bilateral effort from the United States and Japan to develop global clinical trials and address regulatory barriers that may be impediments to timely device approval. The Global Harmonization Task Force (GHTF) was conceived in 1992 in an effort to achieve greater uniformity between national medical device regulatory systems. Since 2012, the GHTF has been replaced by the International Medical Device Regulators Forum.

Abstract  The naphthalene dioxygenase from Sphingomonas CHY-1 exhibits extremely broad substrate specificity toward polycyclic aromatic hydrocarbons (PAHs). In a previous study, the catalytic rates of oxidation of nine PAHs were determined using the purified dioxygenase, but the oxidation products formed from four- to five-ring hydrocarbons were incompletely characterized. Here, we reexamined PAH oxygenation reactions using Escherichia coli recombinant cells overproducing strain CHY-1 dioxygenase. Hydroxylated products generated by the dioxygenase were purified and characterized by means of GC-MS, UV absorbance as well as (1)H- and (13)C-NMR spectroscopy. Fluoranthene was converted to three dihydrodiols, the most abundant of which was identified as cis-7,8-dihydroxy-7,8-dihydrofluoranthene. This diol turned out to be highly unstable, converting to 8-hydroxyfluoranthene by spontaneous dehydration. The dioxygenase also catalyzed dihydroxylations on the C2-C3 and presumably the C1-C2 positions, although at much lower rates. Benz[a]anthracene was converted into three dihydrodiols, hydroxylated in positions C1-C2, C8-C9, and C10-C11, and one bis-cis-dihydrodiol. The latter compound was identified as cis,cis-1,2,10,11-tetrahydroxy-1,2,10,11-tetrahydrobenz[a]anthracene, which resulted from the subsequent dioxygenation of the 1,2- or 10,11-dihydrodiols. Chrysene dioxygenation yielded a single diol identified as cis-3,4-dihydroxy-3,4-dihydrochrysene, which underwent further oxidation to give cis,cis-3,4,9,10 chrysene tetraol. Pyrene was a poor substrate for the CHY-1 dioxygenase and gave a single dihydrodiol hydroxylated on C4 and C5, whereas benzo[a}pyrene was converted to two dihydrodiols, one of which was identified as cis-9,10-dihydrodiol. The selectivity of the dioxygenase is discussed in the light of the known 3D structure of its catalytic component and compared to that of the few enzymes able to attack four- and five-ring PAHs.

Abstract  Previous reports indicated that inhalation of JP-8 aviation turbine fuel is immunosuppressive. However, in some of those studies, the exposure concentrations were underestimated, and percent of test article as vapor or aerosol was not determined. Furthermore, it is unknown whether the observed effects are attributable to the base hydrocarbon fuel (jet fuel kerosene) or to the various fuel additives in jet fuels. The present studies were conducted, in compliance with Good Laboratory Practice (GLP) regulations, to evaluate the effects of jet fuel kerosene on the immune system, in conjunction with an accurate, quantitative characterization of the aerosol and vapor exposure concentrations. Two female rodent species (B6C3F1 mice and Crl:CD rats) were exposed by nose-only inhalation to jet fuel kerosene at targeted concentrations of 0, 500, 1000, or 2000 mg/m(3) for 6 h daily for 28 d. Humoral, cell-mediated, and innate immune functions were subsequently evaluated. No marked effects were observed in either species on body weights, spleen or thymus weights, the T-dependent antibody-forming cell response (plaque assay), or the delayed-type hypersensitivity (DTH) response. With a few exceptions, spleen cell numbers and phenotypes were also unaffected. Natural killer (NK) cell activity in mice was unaffected, while the NK assessment in rats was not usable due to an unusually low response in all groups. These studies demonstrate that inhalation of jet fuel kerosene for 28 d at levels up to 2000 mg/m(3) did not adversely affect the functional immune responses of female mice and rats.

Abstract  Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n=1022) 20-29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of P<.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey-Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects.

Abstract  We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully (13)C-labeled dicarboxylate. Information on the peroxisomal vs mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e., total acetyl-CoA, the acetyl moiety of citrate, C-1+2 of β-hydroxybutyrate, malonyl-CoA and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g., in propionic acidemia and methylmalonic acidemia.

Abstract  Prohibitin (PHB) is a ubiquitously expressed and highly conserved protein that participates in diverse cellular processes, and its functions are linked to a variety of diseases. In the present study, to explore transcriptional activation and signaling pathways involved in PHB regulation in response to sex hormone treatment, we investigated the effects of estrogen (17-β-estradiol, E2) on regulation of PHB in several metabolic tissues from male and female rats. Elevated expression of PHB was prominent in white adipose tissue (WAT) and the liver, and E2 stimulated PHB expression in both ND and HFD-fed rats. To further confirm the expression of PHB which was increased in WAT and the liver, we analyzed PHB expression levels in 3T3-L1 and C9 cells after the treatment of E2. Transcription and protein levels of PHB were dose-dependently increased by E2 treatment in both cell types, supporting our in vivo data. To further evaluate the possible role of E2 in elevation of PHB via estrogen receptors (ER), the potent ER inhibitor fulvestrant was treated to 3T3-L1 and C9 cells. Fulvestrant markedly suppressed both transcription and protein levels of PHB, suggesting that PHB expression in both tissues may be regulated through ERs. GeneMANIA, a predictive web interface, was used to show that Phb is regulated via the intracellular steroid hormone receptor signaling pathway, suggesting a role for ERs in expression of Phb as well as other metabolically important genes. Based on these results, we expect that targeting PHB would be a useful therapeutic approach for treatment of obesity.

Abstract  The product template (PT) domains, specifically in fungal non-reducing polyketide synthases (NR-PKSs), mediate the regioselective cyclization of polyketides dominating the final structures. However, up to date, the systematic knowledge about PT domains has been insufficient. In present study, the relationships between sequences, structures and functions of the PT domains were analyzed with 661 NR-PKS sequences. Based on the phylogenetic analysis, the PT domains were classified into prominent eight groups (I-VIII) corresponding with the representative compounds and cyclization regioselectivity (C2-C7, C4-C9, and C6-C11). Most of the cavity lining residue (CLR) sites in all groups were common, while the regional CLR site mutations resulted in the appearance of finger-like regions with different orientation. The cavity volumes and shapes, even the catalytic dyad positions of PT domains in different groups were corresponding with characteristic cyclization regioselectivity and compound sizes. The conservative residues in PT sequences were responsible for the cyclization functions and the evolution of the key residues resulted in the differentiations of cyclization functions. The above findings may help to better understand the cyclization mechanisms of PT domains and even predict the structural types of the aromatic polyketide products.

Abstract  Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Abstract  Changes in volatile content, as well as associated gene expression and enzyme activity in developing cucumber fruits were investigated in two Cucumis sativus L. lines (No. 26 and No. 14) that differ significantly in fruit flavor. Total volatile, six-carbon (C6) aldehyde, linolenic and linoleic acid content were higher during the early stages, whereas the nine-carbon (C9) aldehyde content was higher during the latter stages in both lines. Expression of C. sativus hydroperoxide lyase (CsHPL) mirrored 13-hydroperoxide lyase (13-HPL) enzyme activity in variety No. 26, whereas CsHPL expression was correlated with 9-hydroperoxide lyase (9-HPL) enzyme activity in cultivar No. 14. 13-HPL activity decreased significantly, while LOX (lipoxygenase) and 9-HPL activity increased along with fruit ripening in both lines, which accounted for the higher C6 and C9 aldehyde content at 0-6 day post anthesis (dpa) and 9-12 dpa, respectively. Volatile compounds from fruits at five developmental stages were analyzed by principal component analysis (PCA), and heatmaps of volatile content, gene expression and enzyme activity were constructed.

Abstract  The volatiles of two different unifloral Palestinian honeys from botanical species Centaurea iberica and Zizyphus spina-christi have been investigated for the first time. They were isolated, identified, and quantitatively analyzed using Headspace Solid-Phase Microextraction and Gas Chromatography Mass Spectrometry (HS-SPME-GCMS) methodology. The resulted total ion current (TIC) chromatographic profiles reflected the uniqueness of each type of honey and therefore the proposed procedure can be used to characterize each kind of honey by revealing the absence or presence of certain volatile constituents.

A total of 18 compounds were seen in Centaurea iberica honey with phenylacetaldehyde, phenylethylalcohol, 2-ethyl hexanoic acid, 2,4,6-trimethylphenol and nonanoic acid as the principal components, whereas 25 compounds were seen in Zizyphus spina-christi honey with benzaldehyde, phenylacetaldehyde, phenylethylalcohol, benzeneacetonitrile, 2-ethyl hexanoic acid, octanoic acid, 2-methoxy-4-(1-propanol)-6-acetate phenol, nonanoic acid, decanoic acid, 1-hydroxy 2,4,6-trimethylbenzene, and 5-hydroxymethyl-2-furaldehyde as the principal constituents.

Ziziphus spina-christi honey was found to have two unequivocal potential markers: phenylacetonitrile and 5-hydroxymethyl-2-furancarboxaldehyde, while Centaurea Italica honey has only one representative floral origin marker compound: the 2,4,6-trimethylphenol.

Abstract  A Gram-staining-negative, strictly aerobic bacterial strain, designated SM-6T, was isolated from a sea tidal flat of the Dangjin bay, South Korea. Strain SM-6T was able to degrade a broad range of aliphatic hydrocarbons. Cells were catalase- and oxidase-positive and non-motile rods. Growth of strain SM-6T was observed at 10-37 C (optimum, 20-25 C), at pH 5.5-9.0 (optimum, pH 6.5-7.5) and in the presence of 0-10 % (w/v) NaCl (optimum, 2-3 %). The only isoprenoid quinone detected was ubiquinone-8 (Q-8). C17:1 ω8c, C11:0 3-OH, summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c), C9:0 3-OH and C10:0 3-OH were observed as the major cellular fatty acids and phosphatidylethanolamine, phosphatidylglycerol and four unidentified lipids were detected as polar lipids. The G+C content of the genomic DNA was 47.5 mol%. Strain SM-6T was most closely related to Pseudomaricurvus alkylphenolicus KU41GT (95.5 %), Maricurvus nonylphenolicus KU41ET (94.4 %) and Pseudoteredinibacter isoporae SW-11T (94.3 %) based on 16S rRNA gene sequences and phylogenetic analyses showed that strain SM-6T formed a phyletic lineage distinct from the closely related genera. On the basis of phenotypic, chemotaxonomic and molecular features, strain SM-6T represents a novel genus and species of the order Alteromonadales in the class Gammaproteobacteria, for which name Aestuariicella hydrocarbonica gen. nov., sp. nov. is proposed. The type strain is SM-6T (= KACC 18121T = JCM 30134T).

Abstract  Primordial germ cells (PGCs) are progenitors of the germ cell lineage, giving rise to either spermatogonia or oogonia after the completion of gonadal differentiation. Currently, there is little information on the mechanism of PGCs migration leading to the formation of the primordial gonad in perciform fish. Yellowtail kingfish (Seriola lalandi) (YTK) (order Perciforms) inhabit tropical and temperate waters in the southern hemisphere. Fundamental details into the molecular basis of larval development in this species can be easily studied in Australia, as they are commercially cultured and readily available. In this study, histological analysis of YTK larvae revealed critical time points for the migration of PGCs to the genital ridge, resulting in the subsequent development of the primordial gonad. In YTK larvae at 3, 5, 7 and 10 days post hatch (DPH), PGCs were not yet enclosed by somatic cells, indicating the primordial gonad had not yet started to form. While at 15, 18 and 20 DPH PGCs had already settled at the genital ridge and started to become enclosed by somatic cells indicating the primordial gonad had started to develop. A higher number of PGCs were observed in the larvae at 15 and 18 DPH indicating PGCs proliferation, which corresponds with them becoming enclosed by the somatic cells. Directional migration of PGCs toward the genital ridge is a critical event in the subsequent development of a gonad. In zebrafish, mouse and chicken, stromal-cell derived factor (SDF1) signalling is one of the key molecules for PGC migration. We subsequently isolated from YTK the SDF1 (Slal-SDF1) gene, which encodes for a 98-residue precursor protein with a signal peptide at the N-terminus. There is spatial conservation between fish species of four cysteine residues at positions C9, C11, C34 and C49, expected to form disulphide bonds and stabilize the SDF structure. In YTK, Slal-SDF1 gene expression analyses shows that this gene is expressed in larvae from 1 to 22 DPH and demonstrates distinct spatial localisation in the larvae at 7 DPH. These results provide a platform for further studies into the molecular machinery of PGC migration in yellowtail kingfish, as well as other perciform fish species.

Abstract  Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Abstract  High-density lipoproteins (HDLs) are atheroprotective because of their role in reverse cholesterol transport. The intestine is involved in this process because it synthesizes HDL, removes cholesterol from plasma and excretes it into the lumen. We investigated the role of selected dietary fatty acids on intestinal cholesterol uptake and HDL functionality. Caco-2 monolayers grown on Transwells were supplemented with either palmitic, palmitoleic, oleic, linoleic, docosahexaenoic, eicosapentaenoic, arachidonic or conjugated linoleic acids (CLAs): c9,t11-CLA; t9,t11-CLA; c10,t12-CLA. Cells synthesized HDL in the basolateral compartment for 24h in the absence or presence of an antibody to SR-BI (aSR-BI), which inhibits its interaction with HDL. Free cholesterol (FC) accumulated to a greater extent in the presence than in the absence of aSR-BI, indicating net uptake of FC by SR-BI. Uptake's efficiency was significantly decreased when cells were treated with c9,t11-CLA relative to the other fatty acids. These differences were associated with lower HDL functionality, since neither SR-BI protein expression nor expression and alternative splicing of other genes involved lipid metabolism were affected. Only INSIG2 expression was decreased, with no increase of its target genes. Increasing pre-β-HDL synthesis, by inducing ABCA1 and adding APOA1, resulted in reduced uptake of FC by SR-BI after c9,t11-CLA treatment, indicating reduced functionality of pre-β-HDL. Conversely, treatment with c9,t11-CLA resulted in a greater uptake of FC and esterified cholesterol from mature HDL. Therefore, Caco-2 monolayers administered c9,t11-CLA produced a nonfunctional pre-β-HDL but took up cholesterol more efficiently via SR-BI from mature HDL.

Abstract  Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.

Abstract  Alkanes are attractive fuels for fuel cells due to their high energy density, but their use has not transitioned to biofuel cells. This paper discusses the development of a novel enzyme cascade utilizing alkane monooxygenase (AMO) and alcohol oxidase (AOx) to perform mediated bioelectrocatalytic oxidation of hexane and octane. This was then applied for the bioelectrocatalysis of the jet fuel JP-8, which was tested directly in an enzymatic biofuel cell to evaluate performance. The enzymatic catalysts were shown to be sulfur tolerant and produced power densities up to 3 mW/cm(2) from native JP-8 without desulfurization as opposed to traditional metal catalysts, which require fuel preprocessing.

Abstract  The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.