One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.     

Separation of mitochondria by flow field-flow fractionation for proteomic analysis

Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.     

Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)-dextran countercurrent partitioning

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)-dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two-phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.     

Accumulation mechanism of 111In in malignant tumor (author's transl)]

In order to clarify the accumulation mechanism of 111In in maligant tumor, subcellular distribution of 111In was quantitatively determined. Buffalo rats bearing Morris hepatoma 7316A were injected intraperitoneally 111In-chloride and tumor tissues were removed 24 hours later. Subcellular fractionation of tumor tissues were carried out according to the method of C. de Duve, et al. and radioactivity of each fraction was counted. Most of the total radioactivity was distributed among the soluble, nuclear and lysosomal fractions. On account of its low protein content, the relative specific radioactivity was the highest in the lysosomal fraction. The lysosomal fraction was solubilized gradually and the resultant stepwise release of 111In and acid phosphatase activity were measured. There was a close relationship between them. From these results it was concluded that 111In accumulated especially in the lysosomes. In the electron micrography the tumor lysosomes had already engulfed many foreign materials, so that the lysosomal function would be depressed.     

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.     

Qualitative and quantitative analysis of lipid extracts from rabbit meat by gas chromatography/mass spectrometry.

Analyses of lipid extracts from rabbit meat were carried out by gas chromatography/mass spectrometry (GC/mS) using both electron and chemical ionisation. Ten rabbit carcasses were randomly acquired on the market, from different farms; for each of them muscular tissues from hindleg and breast were analysed. The lipid fractions were extracted, separated and hydrolysed. The fatty acid fractions were derivatised by 2,2-dimethoxypropane. The GC/mS data obtained using electron ionisation (EI) did not allow the complete characterisation of the fatty acid fraction, and for this reason chemical ionisation (CI) was employed using acetonitrile as reactant gas. The data thus obtained show that, for both samples of rabbit tissue, the mean abundance ratio of plasma cholesterol lowering fatty acids and plasma cholesterol elevating fatty acids (PCL/PCE), taken as a parameter describing a desirable lipid uptake, is 2.2 +/- 0.3, significantly higher than the values reported for other meats (0.8-1. 8). These data, together with the high concentration of (n-6) fatty acids, provide a good indication of the high nutritional value of rabbit meat.     

Extraction and fractionation of bile acids and their conjugates using pre-packed microparticulate silica cartridges (Sep-Pak Sil and Bond-Elut C18)

A new method for the extraction of bile acids from human plasma using acetonitrile precipitation of plasma protein and subsequent use of Bond-Elut C18 cartridges is described. After extraction the bile acids can be separated into three fractions: unconjugated, glycine-, and taurine-conjugated, using Sep-Pak SIL cartridges at 4 degrees C, eluting with ethanol--chloroform--water--glacial acetic acid mixtures. These extraction and fractionation procedures were evaluated in terms of recovery, reproducibility and resolution between the fractions. All these parameters were found to be satisfactory. Although the reproducibility of fractionation on Sep-Pak SIL cartridges was found to vary between batches, this did not give rise to significant difficulties. Plasmas from normals and patients with hepatobiliary disease were analysed by capillary gas-liquid chromatography after extraction and fractionation using the procedure described.     

Subcellular distribution of Al, Cu, Mg, Mn and other elements in the human liver

The elemental concentrations and chemical species of Al, Br, Cl, Cu, K, Mg, Mn, Na and I in human liver and its subcellular fractions were studied by several biochemical techniques combined with neutron activation analysis. The highest concentrations of Al, Mg, and I were found in the nuclei, whereas those of Br, Cu, Cl, K, Mn and Na in the cytosol. About 20% of Br, half of Al and most of Cu (78.8%), Mg (65.9%) and Mn (80.6%) remained in the cellulose bags after dialysis of liver homogenate, which were suggested to be bound to macromolecules. K (100%) and more than 95% of Cl and Na were found to be in the dialyzates. Similar results were found in the fractions of nuclei, mitochondria, lysosome and microsome, respectively, after the same treatment. Further study was carried out to elucidate the elemental distribution in the cytosol by ethanol precipitation and by ammonium sulfate fractionation. The results suggested that several kinds of Cu-, Mn- and Mg-bound proteins existed in the cytosol of human liver cells. Received: 3 August 1998 / Revised: 22 September 1998 / Accepted: 26 September 1998     

Effects of UV Radiation and Diet on Polyunsaturated Fatty Acids in the Skin,Ocular Tissue and Dorsal Muscle of Atlantic Salmon (Salmo salar) Held in Outdoor Rearing Tanks

The effect of UV radiation (UVR) on juvenile Atlantic salmon (Salmo salar) was assessed by measuring the fatty acid (FA) profiles of muscle, dorsal and ventral skin, and ocular tissues following 4-month long exposures to four different UVR treatments in outdoor rearing tanks. Fish were fed two different diets (Anchovy- and Herring-oil based) that differed in polyunsaturated fatty acid (PUFA) concentrations. Anchovy-fed salmon had higher concentrations of ALA (alpha-linoleic acid; 18:3n-3), EPA (eicosapentaenoic acid; 20:5n-3) and DPA (docosapentaenoic acid, 22:5n-3) in their muscle tissues than fish fed the Herring feed. Fish subjected to enhanced UVB levels had higher concentrations of LIN (linolenic acid, 18:2n-6) and ALA, total omega-6 FA and SAFA (saturated fatty acids) in their tissues compared with fish in reduced UV treatments. Concentrations of ALA, LIN, GLA (gamma-linolenic acid; 18:3n-6), EPA, PUFA and total FA were higher in ventral skin of fish exposed to enhanced UVB compared with fish in reduced UV treatments. Salmon exposed to reduced UV weighed more per-unit-length than fish exposed to ambient sunlight. The FA profiles suggest that fish exposed to UV radiation were more quiescent than fish in the reduced UV treatments resulting in a buildup of catabolic substrates.     

用中子活化分析法研究25种元素在人体肝脏亚细胞组分中的分布

采用差速离心分离技术与中子活化分析法（ＮＡＡ）相结合的方法分析了人肝２５５中元素,并用标准参考物保证结果的准确性。分析结果表明,大多数元素在各亚细胞组分中并非均匀分布。Ａｓ、Ａｕ、Ｃｏ、Ｉ、Ｍｇ、Ｓｂ、Ｓｅ在细胞核和线粒体浓度最高。Ａｌ、Ｓｓ、Ａｕ、Ｂａ、Ｆｅ、Ｉ、Ｍｇ、Ｍｏ、Ｓｂ、Ｓｃ在细胞核和线粒体浓度最高。Ａｌ、Ａｓ、Ａｕ、Ｂａ、Ｆｅ、Ｉ、Ｌａ、Ｍｇ、Ｍｎ、Ｓｂ、Ｓｅ在胞液中深度最低,生物体     

A Click Cage: Organelle‐Specific Uncaging of Lipid Messengers

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell‐wide concentration changes. Herein, we report on a coumarin‐based photocaging group that can be modified with organelle‐targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.     

A Combination of Visudyne and a Lipid‐anchored Liposomal Formulation of Benzoporphyrin Derivative Enhances Photodynamic Therapy Efficacy in a 3D Model for Ovarian Cancer

A major objective in developing new treatment approaches for lethal tumors is to reduce toxicity to normal tissues while maintaining therapeutic efficacy. Photodynamic therapy (PDT) provides a mechanistically distinct approach to treat tumors without the systemic toxicity of chemotherapy drugs. PDT involves the light‐based activation of a small molecule, a photosensitizer (PS), to generate reactive molecular species (RMS) that are toxic to target tissue. Depending on the PS localization, various cellular and subcellular components can be targeted, causing selective photodamage. It has been shown that targeted lysosomal photodamage followed by, or simultaneous with, mitochondrial photodamage using two different PS results in a considerable enhancement in PDT efficacy. Here, two liposomal formulations of benzoporphyrin derivative (BPD): (1) Visudyne (clinically approved) and (2) an in‐house formulation entrapping a lipid conjugate of BPD are used in combination with direct PS localization to mitochondria, endoplasmic reticulum and lysosomes, enabling simultaneous photodamage to all three organelles using a single wavelength of light. Building on findings by our group, and others, this study demonstrates, for the first time in a 3D model for ovarian cancer, that BPD‐mediated photodestruction of lysosomes and mitochondria/ER significantly enhances PDT efficacy at lower light doses than treatment with either PS formulation alone.     

Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid method was developed for the simultaneous profiling of 29 free fatty acids in plasma using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS). Barium acetate was used as the cationization agent in the positive ion mode for sensitive multiple reaction monitoring (MRM) experiments. The cis- and trans-C18:1 and -C18:2 isomers were baseline-separated using two tandem reversed-phase C18 UPLC columns, while identification of two pairs of positional isomers of C18:3 and C20:3 required isomer-specific product ions, as the analytes were not chromatographically resolved. The assay linearity was greater than three orders of magnitude and correlation coefficients were >0.99; the limits of detections were typically less than 0.2 microM. The method was successfully applied to plasma free fatty acid profiling of samples from volunteers who participated in a randomized crossover study involving the administration of either placebo or fish oil capsules. The results clearly indicate the ability to measure the time profiles of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma for the volunteers given fish oil capsules while the concentrations of the other free fatty acids and the total free fatty acid concentration in plasma remained virtually constant.     

Toroidal Coil Countercurrent Chromatography: A Fast Simple Alternative to Countercurrent Distribution Using Aqueous Two Phase Partition: Principles,Theory, and Apparatus

Abstract

The principles, theoretical basis and equipment for continuous two phase toroidal coil chromatography are described. Rat liver homogenates were subjected to analytical subcellular fractionation by toroidal coil chromatography in a phase mixture of 3.3% (w/w) dextran T500, 5.4% (w/w) poly(ethylene glycol) 6000, 10 mM sodium phosphate-phosphoric acid buffer, pH 7.4, in 0.26 M sucrose containing 0.05 mM Na₂EDTA and 1 mM ethanol. The distribution of organelles, as reflected by their marker enzymes, was compared to that obtained by discrete counter-current partition in a 17 transfer apparatus. Toroidal coil chromatography showed enhanced resolution of certain organelles. In particular, almost complete separation of plasma membrane from endoplasmic reticulum was achieved and some resolution of plasma membrane from lysosomes was obtained. It is concluded that toroidal coil chromatography offers a potentially useful alternative approach to organelle separation techniques.     

Rapid dioxin assessment in fish products by fatty acid pattern recognition

A novel, cost- and time-effective dioxin screening method relying on fatty acid profile was developed for fish products. The method is based on multivariate covariance between fatty acid composition and dioxin. A dioxin range varying from 1.1 to 47.1 ng TEQ-WHO kg fat(-1) was investigated using 64 fish meal samples. An optimal multivariate dioxin prediction model was developed based on reduction from the original 32 to 13 fatty acids, thus increasing the parsimony and the robustness of the model. The model obtained with three partial least squares regression (PLS) components included the following 13 fatty acids: C14:1 n-5, C16:4 n-1, C18:1 n-9, C18:2 n-6, C18:3 n-6, C18:3 n-3, C20:0, C20:1 n-9, C20:4 n-6, C20:3 n-3, C22:1 n-7, C22:6 n-3, C24:1 n-9. Considering the whole investigated dioxin range, the performance of the PLS model based upon full cross-validation yielded a correlation of 0.90 (r(2)) and a prediction error of 3.31 ng PCDD/F TEQ-WHO kg fat(-1). A submodel of samples in the lower dioxin range 1 to 15 ng PCDD/F TEQ-WHO kg fat(-1) returned a r(2) of 0.88 and an error of 1.85 ng PCDD/F TEQ-WHO kg fat(-1).     

Low n-6/n-3 Gestation and Lactation Diets Influence Early Performance,Muscle and Adipose Polyunsaturated Fatty Acid Content and Deposition,and Relative Abundance of Proteins in Suckling Piglets

Elevated omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) ratios in swine diets can potentially impose a higher risk of inflammatory and metabolic diseases in swine. A low ratio between the two omega PUFAs has beneficial effects on sows’ and piglets’ production performance and immunity status. At present, there are few studies on how sow nutrition directly affects the protein and fat deposition in suckling piglets. Two groups of sows were fed diets with high or low n-6/n-3 polyunsaturated ratios of 13:1 (SOY) and 4:1 (LIN), respectively, during gestation and lactation. Longissimus dorsi muscle and adipose tissue from newborn piglets, nourished only with sow’s milk, were subjected to fatty acid profiling by gas chromatography–mass spectrometry (GC-MS) and to proteomics assays based on nano-liquid chromatography coupled to high-resolution tandem mass spectrometry (nLC-HRMS). Fatty acid profiles on both muscle and adipose tissues resembled the magnitude of the differences between fatty acid across diets. Proteomic analysis revealed overabundance of 4 muscle and 11 adipose tissue proteins in SOY compared to LIN in both piglet tissues. The detected overabundance of haptoglobin, an acute-phase protein, and the stimulation of protein-coding genes and proteins related to the innate immune response and acute inflammatory response could be associated with the pro-inflammatory role of n-6 PUFAs.     

Supplemental Feeding of Laying Hens with Wood Vinegar to Decrease the Ratio of n-6 to n-3 Fatty Acids in Eggs

A balanced ratio of fatty acids n-6 to n-3 in chicken eggs is important for health and to help prevent and manage obesity and other diseases. Traditionally, fish oil or flax seed has been utilized as feed additives to decrease the ratio of n-6 to n-3(n-6:n-3) fatty acids in eggs. The hull of spina date seed(HSDS) is a common agricultural waste product in China, from which wood vinegar(HSDSWV) may be derived. This study evaluated HSDSWV as a supplement in hen feeds to improve the quality of eggs and decrease the ratio of fatty acids n-6:n-3. HSDSWV was obtained via carbonization, and refined. Six concentrations(nil to 0.5%) of HSDSWV were prepared and fed to 6 hen groups, respectively, for 50 d. The fatty acids of the hen's egg yolks were analyzed by gas chromatography/electron ionization-mass spectrometry(GC/EI-MS) in the selected ion monitoring(SIM) mode. The 0.2% HSDSWV resulted in the best egg yolk quality, with a lower percentage of linoleic acid(C18:2n6) and higher percentages of cis-5,8,11,14,17-eicosapentaenoic acid(C20:5n3) and cis-4,7,10,13,16,19-docosahexaenoic acid(C22:6n3), and thus a lower n-6:n-3 ratio compared with the other HSDSWV concentrations. In addition, the eggs contained higher levels of yolk fat and egg yolk than the controls did. In conclusion, to modify the fatty acid composition of hens' eggs and obtain a balanced ratio of n-6:n-3, 0.2% HSDSWV may be considered suitable as a dietary supplement in hens' feed.     

SUBCELLULAR LOCALIZATION OF PORPHYRINS USING CONFOCAL LASER SCANNING MICROSCOPY

The in vitro subcellular distribution patterns of 10 porphyrins, varying in hydrophobicity and charge, were studied using confocal laser scanning microscopy on two cell lines (V79 and C6 glioma cells) for incubation times up to 24 h. All of the porphyrins were taken up rapidly by both cell lines and distinct classes of subcellular distribution patterns were observed: general cytoplasmic staining; localization in lysosomes (usually associated with general cytoplasmic staining); localization in mitochondria (and general cytoplasmic staining); localization in mitochondria with subsequent uptake into lysosomes. Structure-localization relationships which have emerged are that porphyrins with dominantly cationic side chains localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.     

Recent Advances of AIEgens for Targeted Imaging of Subcellular Organelles

Fluorescence imaging based on luminogens with aggregation-induced emission(AIE)effect has drawn great attention in recent two decades,due to their superior advantages to overcome the technical difficulties.Thus,the AIE-active bioprobes with targeted ability at the subcellular level have been widely investigated to visualize the subcellular structures and monitor the biological processes.Considering the very rapid developments and the significance of selective imaging of subcellular structures,we summarize the recent two-year achievements about the AIEgens for targeted imaging of subcellular organelles including nuclei,membranes,lipid droplets(LDs),endoplasmic reticulum(ER),lysosomes,mitochondria and cytoplasm.The designed protocols and advantages of AIEgens,their mechanisms for targeted staining at organelles and the imaging performance are discussed.These AIE bioprobes exhibit great potentials for early diagnosis and therapeutics of diseases that related to subcellular organelles.Finally,the perspectives about AIEgens for these applications are also discussed.     

Antibacterial study and fatty acid analysis of lipids of the sponge <Emphasis Type="Italic">Myrmekioderma granulata</Emphasis>

The composition of the lipophilic extract of the sponge Myrmekioderma granulata (Esper) collected from 13 m depth of the Bay of Bengal of the Orissa coast was investigated. Fatty acids as well as volatiles and sterols were identified. 4,8,12-Trimethyltridecanoic acid was identified for the first time along with the important PUFAs such as linoleic acid (n-6, C18:2), dihomo-γ-linolenic acid (n-6, C20:3), 5,8,11,14-eicosatetraenoic acid (n-3, C20:4), and 5,8,11,14,17-eicosapentaenoic acid (EPA) (n-3, C20:5) from this species. The branched polyunsaturated fatty acids like br-C26:2, 25-methyl-5,9-heptacosadienoic acid and 24-methyl-5,9heptacosadienoic acid were also identified by GC-MS. The lipid extract exhibited limited activity against different pathogens.