Effect Of Isolation Procedures Research Articles

Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC–MS/MS

Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC–MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2–3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration–effects relationship.

Fragmentation Pathways of Polycyclic Polyisoprenylated Benzophenones and Degradation Profile of Nemorosone by Multiple-Stage Tandem Mass Spectrometry

Nemorosone is a polycyclic polyisoprenylated benzophenone (PPBs) with strong cytotoxic activity. It is the major constituent of Clusia rosea floral resin and brown Cuban propolis. Other PPBs found in Cuban propolis are oxidized and cyclized derivatives of nemorosone. The instability of PPBs carrying an enolizable 1,3-diketone system has been suggested, and the elucidation of this aspect is very fundamental for the evaluation of their biologic activity. Electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)) was employed to shed light on the origin of these derivatives of nemorosone and to define the stability of this natural product. For this purpose, we initially performed MS(n) experiments on seven related PPBs to obtain useful information for structural characterization of this class of compounds and to identify the degradation products of nemorosone. The proposed fragmentation pathways, supported by exact mass measurements, allowed the nature of side chains on the bicyclo core and the type and position of their modifications to be established. In a second part, the degradation profile of nemorosone under different conditions was investigated to assess the possible effects of isolation procedures, climatic, and storage conditions on its stability. Our results reveal that nemorosone undergoes rapid degradation in n-hexane and chloroform solutions. The degradation products, identified by HPLC-ESI/MS(n) and NMR, are identical to derivatives of nemorosone previously isolated from propolis and plants. Thus, these PPBs are artefacts formed predominantly during the extraction and purification procedures.

Protein kinase C distribution and translocation in rat myocardium: Methodological considerations

Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass–glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (α, ɛ) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. Methods: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (α, ɛ) distributions were assessed by differential centrifugation and Western blotting. Results: Following 100 000 × g centrifugation of the homogenate, resuspension of the pellet (P 1) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (α, ɛ) such that PKCɛ comigrated with a nonspecific band. Resuspension of P 1 in Triton X-100 (TX) did not affect mobility but decreased P 1 PKC (α, ɛ) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (α, ɛ) were also noted in soluble and P 2 (supernatant of 100 000 × g centrifugation of P 1) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P 2 fraction also revealed Polytron-dependent decreases (47% vs. glass–glass tissue grinder; p<0.05) in PKCɛ following an initial 1000 × g centrifugation and an increased PMA-dependent translocation of PKC (α, ɛ; 2.4-fold and 1.6-fold, respectively, vs. P 1; p<0.05). Discussion: Taken together, these results suggest that PKC isolation procedures should be carefully considered when designing or comparing LV PKC studies due to the potential effects isolation may have on PKC distribution and translocation.

Effect of isolation procedures on the molecular composition and physical properties of Eucheuma cottonii carrageenan

Direct treatment of Eucheuma cottonii with hot potassium hydroxide leads to a solid residue (alkali treated weed) which was found to contain predominantly carrageenan and cellulose. Following dispersion and heating in water, separation into polymer-containing solution and an insoluble particulate phase was readily achieved. Chemical analysis of solubilized polymers showed that the predominant carrageenan present in native weed was a mu/kappa-carrageenan which was readily converted into kappa with hot alkali. The particulate phase from both materials was predominantly cellulose; characterization by X-ray diffraction showed the presence of partial cellulose type I crystallinity. Solid state 13C-NMR spectra showed that this crystalline fraction was ~53% of the total cellulose and was comprised of ~40% type I α and ~60% type I β . The cellulosic part seemed unaffected by the hot alkali treatment. A carrageenan type polysaccharide was found associated with the cellulosic fraction; which underwent the same conversion as the mu/kappa carrageenan on alkali treatment. Further refining of solubilized carrageenan by isopropyl alcohol precipitation, gel pressing or freeze-drying, resulted in only minor differences in chemical composition. Hot solution viscosity and gelation properties in the presence of potassium chloride were measured showing no significant differences in gelling behaviour between alkali treated weed and the different carrageenan isolates but a higher solution viscosity for the alkali treated weed.

Influence of hemicelluloses on the aggregation patterns of bacterial cellulose

Cellulose from the bacteriumAcetobacter xylinum was used as a model system for investigating the influence of other cell wall polysaccharides on the aggregation of cellulose. The patterns of aggregation of the bacterial cellulose were modified when the cellulose was produced in the presence of hemicellulose-like saccharides. The celluloses were found to be more like the Iβ-type found in higher plant celluloses than the Iα-type in the control bacterial celluloses. The effects of isolation procedures on structure were also explored. It was found that the structures of isolated celluloses were influenced by the procedures used in isolation.

Effect of isolation procedure on molecular weight distribution and monosaccharide composition of cotton stalk lignins

Neutral detergent fiber (NDF) of cotton stalks (CS) was ball milled for 7, 14, 21 and 28 days in a porcelain rotary ball mill, hydrolyzed by a cellulase for 4 days and lignin extracted from it by either dioxane:H 2O or 1 M NaOH. The effects of ball-milling duration (BMD) and extraction procedure on yield, molecular weight distribution and carbohydrate content of lignins were investigated in this study. The dioxane system extracted 25% of the permanganate lignin following 28 days of BM; the major increment, from 10.6 to 20% was observed between the 7th and 14th days of BM. The alkali system reached its yield peak at 14 days of BM at which time 56% of the permanganate lignin had been extracted. The molecular weight distribution pattern of the dioxane lignins (DL) was constant irrespective of BMD. The alkali system had probably targeted larger structures of lignin since the proportion of 23–123 kDa molecules in alkali lignins (AL) was two to three times higher than in DLs. The 14 day BM AL contained the largest proportion of 23–123 kDa lignins, had the highest “weight average molecular weight” (MWt) (17.3 kDa) and was the most dispersive preparation. Both (DL and AL) preparations were low in carbohydrates (5–6 g per 100 g lignin) with xylose and uronic acid as the main monosaccharide components. The high level of uronic acid in the AL preparation indicates that these isolates originated at least partly from the middle lamella area.

Effect of Isolation Procedure on Molecular Weight Distribution of Wheat Straw Lignins

Neutral detergent fiber (NDF) of wheat straw (WS) war ball-milled for 7, 14, 21, and 28 days in a porcelain rotary ball-mill and hydrolyzed by a cellulare for 4 days, and the residue was used for lignin extraction by either diorane or 1 M NaOH. The effects of ball-milling duration (BMD) and extraction procedure on lignin yield and its high-performance size exclusion chromatography (HPSEC) features were examined in this study. Optimal BMD was 14 days, at which 80% and 100% of the permanganate lignin has been extracted by the dioxane and alkali systems, respectively. At a BMD of 14 days, carbohydrate content war 6% and 15% of the alkali lignin (AL) and diorane lignin (DL), respectively

Neutrophil activation during cell separation procedures

Leucocytes labelled with 111In or 99Tcm are used as diagnostic agents for detecting sites of infection by scintigraphy. Before radiolabelling is performed, leucocytes are isolated from whole blood. The effect of isolation procedures on neutrophil activation has been studied by measuring the neutrophil elastase produced during incubation of whole blood with three erythrocyte sedimentation agents (dextran 70, hydroxyethyl starch and methylcellulose) and two density gradient media (Percoll and Mono-Poly Resolving Medium). Neutrophil elastase was measured using a standard radioimmunoassay. At 21 degrees C, dextran caused no elastase release while hydroxyethyl starch and methylcellulose induced significant release (p = 0.01 and p less than 0.01 resp.). All three agents caused significant elastase release at 37 degrees C. When whole blood was incubated with Percoll and Mono-Poly Resolving Medium, no release of neutrophil elastase was observed. These results show that neither density gradient medium induces neutrophil activation but that certain erythrocyte sedimentation agents do. Of the three sedimentation agents investigated, dextran is the agent of choice if neutrophil activation is to be minimized.

Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria. Effect of isolation procedure and mitochondrial delta pH.

In order to study branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria, an improved procedure was developed for isolating muscle mitochondria. The procedure uses the protease Nagase in mannitol sucrose media (Procedure A). These mitochondria exhibited high rates of oxygen consumption, good respiratory control ratios, and improved rates of branched chain alpha-keto acid oxidation. At 20 microM [1-14C]alpha-ketoisovalerate (KIV), rates were 1.99 +/- 0.09 nmol/mg of mitochondrial protein/min versus 0.85 +/- 0.02 in mitochondria prepared in electrolyte media without Nagase treatment (Procedure B). The apparent kinetic constants for KIV and alpha-ketoisocaproate (KIC) oxidation were determined. In the presence of ATP, the Vmax and K0.5 for KIV were 17.7 +/- 2.5 nmol/mg of mitochondrial protein/min and 82 microM, respectively. The K0.5 for KIV was at least 2-fold higher than for KIC as were apparent Vmax values. Branched chain alpha-keto acid oxidative decarboxylation in skeletal muscle mitochondria was compared to the activity in mitochondria isolated from liver, heart, and kidney. Rates of KIV and KIC oxidative decarboxylation were highest in heart mitochondria and quite similar in skeletal muscle, liver, and kidney mitochondria. It is the low mitochondrial content of mixed skeletal muscle, not the specific activity of the branched chain alpha-keto acid dehydrogenase, that limits muscle oxidative capacity. The data also indicate that the total activity in muscle has been routinely underestimated. Addition of ATP which increased the matrix pH (increases delta pH) stimulated the rate of oxidative decarboxylation of branched chain alpha-keto acids. On the other hand, addition of uncoupler which decreased the delta pH inhibited the rate of oxidation. Nigericin in low K+ medium inhibited oxidation to about the same degree as uncoupler, while addition of valinomycin in high K+ medium, which will decrease the electrical potential, had little effect on oxidation rates. Transport of branched chain alpha-keto acids should be sensitive to the mitochondrial pH gradient. Hence, the effects of ATP and the mitochondrial inhibitors on rates of branched chain alpha-keto acid oxidation suggest that mitochondrial transport may be partially rate-controlling for oxidation at physiological concentrations of the branched chain alpha-keto acids.

Gap junction structures. VI. Variation and conservation in connexon conformation and packing

Correlation of structural changes in isolated gap junctions with the mechanism of channel gating is complicated by the effects of isolation procedures and the lack of a direct functional assay. The effect of variations in the isolation procedure are examined by comparison of the structures of gap junctions isolated by different protocols. X-ray diffraction data from over two hundren specimens are compared to provide a basis for identification of invariant aspects of the connexon structure and variable properties related either to functional switching or experimental modifications. We discuss the relationship between subunit tilt, lattice symmetry and packing, and membrane curvature and demonstrate that membrane curvature may be a natural consequence of the structure of the connexons and the patterns of interactions between them.