Double bonds of unsaturated fatty acids differentially regulate mitochondrial cardiolipin remodeling.

BACKGROUND: Supplemented fatty acids can incorporate into cardiolipin (CL) and affect its remodeling. The change in CL species may alter the mitochondrial membrane composition, potentially disturbing the mitochondrial structure and function during inflammation. METHOD: To investigate the effect of the unsaturation of fatty acids on CL, we supplemented macrophage-like RAW264.7 cells with 18-carbon unsaturated fatty acids including oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), γ-linolenic acid (GLA, 18:3), and stearidonic acid (SDA, 18:4). Mitochondrial changes in CL were measured through mass spectrometry. RESULT: Our data indicated that OA(18:1) was the most efficient fatty acid that incorporated into CL, forming symmetrical CL without fatty acid elongation and desaturation. In addition, LA(18:2) and ALA(18:3) were further elongated before incorporation, significantly increasing the number of double bonds and the chain length of CL. GLA and SDA were not optimal substrates for remodeling enzymes. The findings of RT-qPCR experiments revealed that none of these changes in CL occurred through the regulation of CL remodeling- or synthesis-related genes. The fatty acid desaturase and transportation genes-Fads2 and Cpt1a, respectively-were differentially regulated by the supplementation of five unsaturated 18-carbon fatty acids. CONCLUSIONS: The process of fatty acid incorporation to CL was regulated by the fatty acid desaturation and transportation into mitochondria in macrophage. The double bonds of fatty acids significantly affect the incorporation process and preference. Intact OA(18:1) was incorporated to CL; LA(18:2) and ALA(18:3) were desaturated and elongated to long chain fatty acid before the incorporation; GLA(18:3) and SDA(18:4) were unfavorable for the CL incorporation.

Identifying Specific and Differentially Linked Glycosyl Residues in Mammalian Glycans by Targeted LC-MS Analysis.

Glycans, which are widespread in nature, consist of a large number of monosaccharides linked via glycosidic bonds. Due to the complex nature of glycan structures on glycoproteins, assessing the configuration and positions of the glycosidic linkages of a glycan is a subject of considerable interest. In this study, a method for accomplishing this using partially O-methylated alditols (PMAs) from glycans combined with LC-MS analysis is reported. N-Glycans were first per-methylated with methyl iodide, and the levels of methylation were further confirmed by MALDI-TOF. PMAs were then produced via complete hydrolysis and reduction. PMAs derived from Fetuin N-glycan and Lewisa antigen carbohydrates were successfully detected by LC-MS analysis. This analysis can be performed without the need for an additional derivatization step for GC analysis, and should be suitable for use in conjunction with a LC-MS-based analysis platform.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

Correlation between pigmentation and larval settlement deterrence by Pseudoalteromonas sp. sf57.

The red-pigmented marine bacterium Pseudoalteromonas sp. sf57 forms a biofilm that deters larval settlement of the tube-building polychaete Hydroides elegans. To investigate the correlation between pigmentation and larval settlement deterrence, mutants of sf57 with deficient or altered pigmentation were generated by transposon mutagenesis. Five groups of pigmented mutants were obtained, viz. white, yellow, pink, dark red, and white-to-red. The white mutant WM1, which exhibited a substantial increase in bacterial density in the biofilm, became inductive to larval settlement. The other mutants that showed a lesser increase in bacterial density in their biofilms either retained their deterrence or induced higher larval settlement rates, but did not become inductive strains. Analysis of the disrupted genes in these mutants suggests that the type II secretion pathway, the LysR transcriptional regulator, NAD(P)-binding proteins, exonuclease, pyruvate metabolism, flagella assembly, and cell membrane processes may play a role in the regulation of pigmentation in sf57.

Bacterial community succession and chemical profiles of subtidal biofilms in relation to larval settlement of the polychaete Hydroides elegans.

Earlier studies have shown that biofilms can mediate the larval settlement of the polychaete Hydroides elegans and that changes in the bacterial community structure and density of biofilms often alter the larval settlement response. However, the chemical cues that mediate this response remain unknown. In this study, both successional changes in the bacterial community structure and the chemical profiles of subtidal biofilms are described and related to the larval settlement response. Multispecies biofilms were developed on polystyrene Petri dishes and granite rock in the subtidal zone over a period of 20 days. The effects of the substratum and age on the bacterial community structure and chemical profiles of the biofilms were evaluated with two molecular methods (microarray (PhyloChip) and denaturing gradient gel electrophoresis) and with gas chromatography-mass spectrometry, respectively. Both age and substratum altered the bacterial community structures and chemical profiles of the biofilms. Age had a greater effect in shaping the bacterial community structure than did the substratum. In contrast, the type of substratum more strongly affected the chemical profile. Extracts of biofilms of different ages, which developed on different substrata, were tested for the settlement of H. elegans larvae. The extracts induced larval settlement in a biofilm-age-dependent manner, and extracts originating from different substrata of the same age showed no differences in larval settlement. Our results suggest that the larval settlement response cannot be predicted by the overall chemical composition of the biofilm alone.

Analysis of RNA polymerase beta subunit (rpoB) gene sequences for the discriminative power of marine Vibrio species.

In the present study, we sequenced the RNA polymerase beta subunit (rpoB) gene of marine Vibrio species and assessed its discriminative power in identifying vibrios. Both the rpoB and 16S rRNA sequences of 29 phenotypically different Vibrio strains isolated from coastal waters were determined. Molecular and phylogenetic comparisons of the sequences of these two genes classified the 29 strains into 11 different species. The resolution of the Vibrio spp. on the rpoB phylogenetic tree was approximately three times greater than that on the 16S rRNA phylogenetic tree. Moreover, by comparing the rpoB sequences of 98 marine gamma-Proteobacteria, including 38 marine Vibrio species, Vibrio-specific primers were developed to amplify a 730-bp fragment of the rpoB gene. Using these primers, we successfully detected Vibrio signals in environmental samples and determined their relative abundances via comparisons with known standards. This rpoB-targeting polymerase chain reaction assay can be used efficiently to monitor relative Vibrio abundance in marine waters.

Evidence for the dynamics of Acyl homoserine lactone and AHL-producing bacteria during subtidal biofilm formation.

The quorum sensing signals-acyl homoserine lactones (AHLs) were directly detected in 1-9-day-old subtidal biofilms developed in a coastal fish farm by using AHL reporter strains and gas chromatography-mass spectrometry. Both methods showed that the AHL molecules and/or AHL-producing bacterial community were dynamic during biofilm development, with dominant AHLs changed from short-chain to long-chain AHLs. Terminal restriction fragment length polymorphism analysis of the bacterial 16S rRNA genes derived from subtidal biofilms of different ages was compared to that of the 21 AHL-producing bacteria isolated from the same batch of subtidal biofilms. All terminal restriction fragments (TRFs) generated from AHL-producing bacteria matched with the dominant TRFs derived from the biofilm bacterial community samples. Particularly, the TRFs of all AHL-producing Vibrio spp. matched with the TRFs that were dominant only in 1-day-old biofilm, suggesting that AHL-producing vibrios were one of the pioneer groups during subtidal biofilm formation. We reported here for the first time the dynamics of AHLs and AHL-producing bacteria during the formation of a subtidal biofilm.

Effect of biofilm formation by Pseudoalteromonas spongiae on induction of larval settlement of the polychaete Hydroides elegans.

The effects of culture conditions and chloramphenicol treatment on the induction of the marine bacterium Pseudoalteromonas spongiae to larval settlement of Hydroides elegans were investigated. The results showed that P. spongiae cells grown in the medium containing both yeast extract and peptone (YP-grown P. spongiae) was highly inductive to larval settlement, whereas P. spongiae cells grown in the medium containing only peptone (P-grown P. spongiae) or YP-grown P. spongiae cells treated with chloramphenicol at the onset of biofilm development (YPC-grown P. spongiae) did not induce larval settlement. Analysis of biofilm formation, biofilm structure, and the surface protein profile indicated that only the induction-capable YP-grown P. spongiae formed a well-developed biofilm, while the P-grown P. spongiae and the YPC-grown P. spongiae did not. We report here for the first time that bacterial biofilm formation was associated with its induction of larval settlement.

Presence of acyl-homoserine lactone in subtidal biofilm and the implication in larval behavioral response in the polychaete Hydroides elegans.

Quorum sensing (QS) signals have been considered to play important roles in biofilm development and in the attractiveness of biofilms to higher organisms in marine ecosystem. In this study, bacterial QS signalsacylated homoserine lactone derivatives (AHLs) were detected in 2-, 4-, and 6-day-old subtidal biofilms by using AHLs reporter strains. N-dodecanoyl-homoserine lactone (C12-HSL) was identified in 6-day-old biofilm at a concentration of 9.04 microg cm(-minus;2) (3.36 mmol l(-minus;1)). To investigate the possible role of AHLs in the consequent eventlarval settlement of the polychaete Hydroides elegans onto subtidal biofilmsseven biofilm-derived bacteria that effectively induced larval settlement of H. elegans, were screened for AHL production. One of them, the Vibrio sp. UST950701-007, produced N-hexanoyl-homoserine lactone (C6-HSL). Larval settlement bioassay showed that C6-HSL, C12-HSL, and 3-oxo-octanoyl-homoserine lactone (3-oxo-C8-HLS) at certain concentrations induced some initial larval settlement behaviors such as reducing swimming speed, crawling on the bottom. However, these AHLs did not effectively induce larval settlement in comparison to the effective settlement inducer 3-isobutyl-1-methylxanthine. The possible chemokinetic mechanism and indirect effects of AHLs on larval settlement are suggested.

[Uptake of nickel from industrial wastewater by genetically engineered Escherichia coli JM109].

Heavy metal wastewater poses a serious threat to the environment. In comparison to the existing methods of chemical precipitation, ion exchange and carbon adsorption, biosorption is an attractive alternative for the recovery of heavy metals from industrial effluents. However, nickel ion, different from other heavy metal ions, is a more recalcitrant pollutant and has low affinity to many metal tolerant microorganisms. In this study, Escherichia coli JM109 was genetically engineered to simultaneously express a Ni2+ transport system (the product of nixA gene) andoverexpress metallothionein (MT). NixA protein has a high affinity for Ni2+, and metallothioneins (MTs) are capable of binding a variety of heavy metals including Ni2+ . The Ni2+ bioaccumulation performance of the genetically engineered E. coli JM109 was evaluated. Time-course test showed that the bioaccumulation rate was rapid, and 95% of the accumulation was achieved within the first 10 minutes. The maximum Ni2+ bioaccumulation by genetically engineered E. coli cells was dramatically increased from 1.54 mg/g to 10.11mg/g, a more than five-fold increase than that of the original E. coli strain. The isotherm was of Langmuir type. Within the tested pH range (pH 4-10), the engineered cells displayed more resistance to pH variation, retaining up to 80% of the Ni2+ binding capacity at pH 4, while the original E. coli host cells lost 80% of Ni2+ binding capacity at pH 4. The presence of Na+ and Ca2+ affected Ni2+ bioaccumulation, but the effects were not serious, as 71% and 66% of the Ni2+ binding capacities were retained respectively at the concentrations of 1000 mg/L Na+ and 1000 mg/L Ca2+ . However, Mg2+ exerted a severe adverse effect on Ni2+ bioaccumulation, 83% of Ni2+ accumulating capacity was lost when Mg2+ concentration reached 200 mg/L. The effects of different kinds of heavy metals on Ni2+ accumulating were different. The genetically engineered E. coli cell lost less than 45% of its Ni2+ bioaccumulation activity in the presence of 50 mg/L lead or cadmium, 66% in the presence of 25mg/L mercury and 84% in the presence of 40 mg/L copper. The presence of glucose did not improve Ni2+ uptake. Our study suggests that the genetically engineered E. coli JM109 has potential application for effective and efficient recovery of nickel from aqueous solutions.