Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes.

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.

Apoptosis regulation at the mitochondrial outer membrane.

Mitochondria play a critical role in apoptosis, or programmed cell death, by releasing apoptogenic factors from the intermembrane space. This process, known as mitochondrial outer membrane permeabilization (MOMP), is tightly regulated by the Bcl-2 family proteins. Pro-apoptotic Bcl-2 family members, Bax and Bak, change their conformation when activated by BH3 domain-only proteins in the family and permeabilize the MOM, whereas pro-survival members inhibit permeabilization. The precise nature of the apoptotic pore in the MOM is unknown, but is probably lipidic. Furthermore, it has been realized that there is another layer of MOMP regulation by a protein factor termed the catalyst in the MOM in order for Bax/Bak to achieve efficient and complete membrane permeabilization. Mitochondrial dynamics do not affect MOMP directly, but seem closely coordinated with MOMP for swift protein efflux from mitochondria. This review will present current views on the molecular mechanisms and regulation of MOMP and conclude with recent developments in clinical applications based on the knowledge gleaned from the investigation.

Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species.

Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified.

Multiple precursor ion scanning of gangliosides and sulfatides with a reversed-phase microfluidic chip and quadrupole time-of-flight mass spectrometry.

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails.

Intakes and breast-milk concentrations of essential fatty acids are low among Bangladeshi women with 24-48-month-old children.

Maternal fat intake and adipose reserves are major sources of PUFA during lactation. The present study examined the cross-sectional relationship between prolonged breast-feeding and maternal BMI, assessed adequacy of fat intake among lactating and non-lactating mothers of children 24-48 months of age and determined breast-milk fatty acid composition. Multi-stage sampling was used to select a representative sample of mothers from two rural districts in Bangladesh (n 474). Dietary data were collected during two non-consecutive 24 h periods via 12 h in-home daytime observations and recall. The National Cancer Institute method for episodically consumed foods was used to estimate usual intake distributions. Breast milk samples were collected from ninety-eight women, and breast-milk fatty acid methyl esters were quantified using GC. Approximately 42 % of lactating v. 26 % of non-lactating mothers were underweight (BMI < 18·5 kg/m2; P = 0·0003). The maternal diet was low in total fat (approximately 8 % of mean total energy) and food sources of PUFA, including oil and animal source foods, resulting in a low estimated mean total consumption of PUFA (5·1 g/d). Almost all women were estimated to consume less than the recommended intake levels for total fat, total PUFA, α-linolenic acid (ALA) and DHA. Median breast-milk linoleic acid (8·5 % weight) and ALA (0·2 %) concentrations were among the lowest reported in the literature, in contrast with arachidonic acid (0·5 %) and DHA (0·3 %) concentrations, which were mid-range. Bangladeshi women in general, and especially those who practise prolonged breast-feeding, may benefit from increased consumption of food sources of PUFA.

Chemo-enzymatic detection of protein isoaspartate using protein isoaspartate methyltransferase and hydrazine trapping.

Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.

Lipidomics and lipid profiling in metabolomics.

PURPOSE OF REVIEW: The field of metabolomics is extending the principles of genomics into cellular and organism metabolism and driving a revolution in lipid biochemistry, physiology and nutrition. Lipids studied using metabolomic approaches - lipidomics - are an ideal subject for metabolomic measurements. RECENT FINDINGS: Lipids are small molecules that share common physical and chemical properties as a class, whose presence and abundance are key to much of metabolic regulation, from subcellular compartments to whole body energy control and signaling. Furthermore, by measuring changes in lipid concentrations, scientists are gaining a more detailed understanding of biochemistry and thus annotating genomes, but also understanding genetic polymorphisms and the postgenetic effects induced by drugs, foods and toxins. SUMMARY: Although in its infancy - there are fewer than 200 total articles on lipidomics and metabolomics focusing on lipids - the field of metabolomics is beginning to deliver on its promise to revolutionize lipid and metabolic disease research.