Nycodenz Density Gradient Centrifugation Research Articles

Analysis of Yeast Peroxisomes via Spatial Proteomics.

Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.

A method for isolating highly purified and active mitochondria from insects

So far, methods that yield the high purity and activity of the isolated mitochondria from insects have not been reported and determined. Here, we develop methods that combine differential centrifugation and discontinuous Nycodenz density gradient centrifugation to isolate highly purified mitochondria from the thorax muscle of insects, and the methods were widely validated across three orders (Coleoptera, Hymenoptera, and Blattaria) covering four insect species using Western blot and transmission electron microscopy (TEM) analysis. The results showed the removal of the residual contamination with nonmitochondrial components such as nucleus, sarcolemma, cytosol, and endoplasmic reticulum. Furthermore, TEM, mitochondria staining, fluorescence detection, and flow cytometry analyses were employed to assess membrane integrity and activity of the isolated mitochondria. The results showed no loss of mitochondria activity/integrity after isolation. In addition, temporal dynamics in activity of the isolated mitochondria under commonly used laboratory temperature (-20 °C, 4 °C, and 25 °C) were respectively detected using a fluorescence microplate reader. The results showed that it should be avoided to store the isolated mitochondria at room temperature, and the mitochondria can meet the requirements of the most downstream experiments when they were stored at −20 °C. Overall, the study presented a method for isolating highly purified and active mitochondria from insects. This study firstly described a high-speed discontinuous density gradient centrifugation-based method that could be widely applied for mitochondria isolation in insects. The present study also provided an example to assess purity and integrity/activity of the isolated mitochondria.

Development of a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method for screening mitochondria-targeted bioactive constituents from complex matrixes: Herbal medicines as a case study

Mitochondria are an important intracellular pharmacological target because damage to this organelle results in a variety of human disorders and because mitochondria are involved in complex processes such as energy generation, apoptosis and lipid metabolism. To expedite the search for natural bioactive compounds targeting mitochondria, we initially developed an efficient mitochondria-based screening method by combining centrifugal ultrafiltration (CU) with liquid chromatography/mass spectrometry (LC/MS), which is called screening method for mitochondria-targeted bioactive constituents (SM-MBC) and is compatible with the search of mitochondria-targeted compounds from complex matrixes such as herbal medicines extracts. Functionally active, structurally intact and pure mitochondria were obtained from rat myocardium using an optimized protocol for mitochondrial isolation comprising organelle release followed by differential and Nycodenz density gradient centrifugation. After evaluating the reliability of the method using thiabendazole (TZ), rotenone (RN), amiodarone (AR) and trimetazidine (TD) as positive controls, this method was successfully applied to screen bioactive constituents from extracts of Polygoni Cuspidati Rhizoma et Radix (PCRR) and Scutellariae Radix (SR). Nineteen active compounds were detected and identified by LC/MS, of which 17 were new mitochondria-targeted compounds. The activity of 9 of the 19 hit compounds was confirmed by in vitro pharmacological trials. These results demonstrate that SM-MBC can be used for the efficient screening of mitochondria-targeted constituents in complex preparations used to treat mitochondrial disorders, such as PCRR and SR. The results may be meaningful for an in-depth understanding of drug mechanism of action and drug discovery from medicinal herbs.

Isolation and number of circulated primordial germ cells (circulated-PGCs) on stages of embryonic development of Gaok chicken

Avian primordial germ cell (PGCs) show a unique migration pathway during early development. During the early embryonic development, as soon as the formation of blood vessels, PGCs enter the circulatory system and migrate to the gonadal primordial. The aim of this study was to examine the number of circulated-PGCs from Gaok chicken at different developmental stages of embryo. One hundred fertile eggs were divided into 5 groups and incubated in a portable incubator at 38 o C and humidity 60%. Hatching was set according to the embryonic development stage between 14-18. The blood collection was done through the dorsal aorta using micropipette under microscope. The collected blood was grouped based on the embryonic stages and placed on a 1.5 ml eppendorf tube which had been filled with 1.000 µl of Calcium and Magnesium-free phosphate buffered saline (PBS -). The PGCs were then purified using nycodenz density gradient centrifugation. The results showed that the average number of circulated-PGCs per embryo from Gaok chicken were significantly affected by the stage of embryonic development (P < 0.05). The number of circulated-PGCs at stages 14, 15, 16, 17 and 18 were 42.8 ± 8.9, 51.0 ± 5.8, 37.6 ± 5.9, 32.8 ± 3.6 and 32.6 ± 3.2, respectively. However, the number of circulated-PGCs was no different between stage of 17 and 18. At Gaok chicken, the number of circulated-PGCs reach the peak at stage 15, it is recommended that collection of PGCs embryonic chicken from blood circulation was the best on stage 15. This information is useful in efficiency production of germline chimera and to preserve PGCs of other Indonesian native chicken. Key Words: Gaok Chicken, PGCs, Embryonic Development Stages

The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

Effects of salvianolic-acid B on the p38MAPK signaling pathway and its transcriptional factor induced by transforming growth factor b1 in activated rat hepatic stellate cells

To investigate the effects of Salvianolic-acid B on p38MAPK signaling pathway and its transcriptional factor activated by Transforming growth factor b1 in rat hepatic stellate cells. Hepatic stellate cells were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method.TGFb1 (10 ng/ml), PD98059(50 mumol/L), SB203580(10 mumol/L) and SA-B (10-6 mol/L) were directly added to the medium of the isolated HSCs. Groups: (1)The detection of total p38, MKK3/6, MEF2A and MEF2C induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group and TGFb1 group. (2)The detection of the phosphorylation of p38, MKK3/6 and a-SMA induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group, TGFb1 group, PD98059 group, PD98059+SA-B group, PD98059+TGFb1 group and SA-B+PD98059+TGFb1 group. (3)The effects of SA-B on activity of MEF2 reporter and collagen a 1(I) reporter induced by TGFb1 in HSC: include mt group, wt group, TGFb1 group, SA-B+TGFb1 group, SA-B group, SB203580+TGFb1 group and SB203580 group. Total and phosphorylated p38 and MKK3/6, MEF2A, MEF2C and a-SMA were assayed by Western blot. HSCs were transfected with either MEF2 or collagen a1(I) luciferase reporter gene by Lipofectamine 2000 transfection method, Cellular extracts were assayed for both MEF2 and collagen a1(I) luciferase activities. Comparisons between groups were performed with Student-Newman-Keuls test. The relative expression level of the phosphorylation of p38 of SA-B group is 0.33+/-0.05,obviously lower than control group(q=7.08, P less than 0.01); SA-B+TGFb1 group is 0.46+/-0.04, obviously lower than TGF b1 group(q=10.45, P less than 0.01); The relative expression level of the phosphorylation of MKK3/6 of SA-B group is 0.11+/-0.07, obviously lower than control group(q=3.944, P less than 0.05); SA-B+TGF b1 group is 0.28+/-0.07, obviously lower than TGFb1 group (q=7.91, P less than 0.01); The relative luciferase activity of MEF2 reporter of SA-B+TGFb1 group and SB203580+TGF b1 group is 2.93+/-0.09 and 2.50+/-0.05 respectively, both obviously lower than TGFb1 group(q=35.35 and 37.2, P less than 0.01); The relative expression level of MEF2C and MEF2A of SA-B group is 15.82+/-0.97 and 13.00+/-0.40 respectively, obviously lower than control group(q is 5.18 and 13.32, both P less than 0.01); SA-B+TGF b1 group is 13.40+/-0.72 and 20.47+/-0.83 respectively, obviously lower than TGFb1 group(q is 43.93 and 12.52,both P less than 0.01); The relative expression level of a-SMA of SA-B+TGFb1 group is 8.76+/-0.44, obviously lower than TGFb1 group(q=20.35, P less than 0.01); SA-B+SB203580+TGFb1 group is only 3.57+/-0.49, obviously lower than TGFb1 group(q=39.78, P less than 0.01); The relative luciferase activity of collagen a1(I) reporter of SA-B+TGF b1 group and SB203580+TGFb1 group is 1.61+/-0.05 and 1.42+/-0.07 respectively, obviously lower than TGFb1 group(q=26.4 and 27.62, both P less than 0.01). SA-B could inhibit activation of HSC induced by TGFb1 through inhibiting p38MAPK signaling pathway in hepatic stellate cells.

Effect of the dimeric bile acid analogue S 0960, a specific inhibitor of the apical sodium-dependent bile salt transporter in the ileum, on the renal handling of taurocholate.

The effect of the dimeric bile acid analogue S 0960 (CAS 142974-51-4), a specific inhibitor of the apical sodium-dependent bile salt transporter (ASBT) in the ileum, on kidney function was studied by clearance experiments in anesthetized rats. Additional experiments were performed on proximal tubular cells freshly isolated from rat kidney cortex and enriched by nycodenz density gradient centrifugation. The clearance studies, which were performed after a 5 h bile duct ligation, revealed a marked rise of the 3H-taurocholate clearance (from 85.4 +/- 15.7 to 371.1 +/- 86.0 microliters/min 100 g b.w., p < 0.05) and a considerable fall of the fractional tubular 3H-taurocholate reabsorption (from 90.2 +/- 1.72 to 68.2 +/- 7.50%, p < 0.05) after S 0960 at a dose of 10 mg/kg i.v. whereas the glomerular filtration rate did not significantly change (from 919 +/- 165 to 1055 +/- 162 microliters/min/100 g b.w.). Isolated proximal tubular cells showed a significant accumulation of 3H-taurocholate. The 3H-taurocholate cell/bath concentration ratio amounted to 3.34 +/- 0.17 at a 3H-taurocholate bath concentration of 3 x 10(-7) mol/l. LiCl (10(-3) mol/l), which is known to inhibit sodium-dependent transport processes in the kidney, markedly diminished cellular 3H-taurocholate uptake (by 65.8%) whereas probenecid (CAS 57-66-9, 10(-4) mol/l), the classical inhibitor of the basolateral organic acid transporter in the kidney, did not significantly affect 3H-taurocholate uptake. This finding indicates that transport of taurocholate by the basolaterally located organic acid transporter is not involved in the uptake process. The kinetic studies revealed an apparent K(m) value of 31 mumol/l and a Vmax value of 6.7 mumol/l cell water/min for tubular 3H-taurocholate uptake. At concentrations > 30 mumol/l S 0960 virtually completely inhibited cellular 3H-taurocholate uptake. 3H-taurocholate uptake was half-maximally inhibited at a S 0960 concentration of 5.8 mumol/l. The results of this functional study are in line with recent molecular evidence that the apical sodium-dependent bile salt transporters in kidney and ileum are identical and demonstrate that S 0960 is a potent inhibitor of the apical sodium-dependent taurocholate transporter in the kidney which augments the renal clearance of 3H-taurocholate. Compounds such as S 0960 may be of special therapeutical value in patients with extrahepatic cholestasis and elevated levels of plasma bile acids.

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction

Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.

Effects of blueberry on the proliferation and activation of hepatic stellate cell and its mechanism

To investigate the effects of blueberry on the proliferation and activation of hepatic stellate cell (HSC) and its mechanism. Rat HSC were isolated by type IV collagenase digestion and Nycodenz density gradient centrifugation. The cultured HSC was incubated at different concentrations of 10% blueberry serum. The 10% DSHX serum was used as positive control and 10% normal rat serum group as control. MTT colorimetric assay was used to detect the HSC proliferation. ColI of culture supernatant was detected by ELISA. The expression of α-SMA in HSC was measured by immunocytochemistry staining while the expressions of Nrf2 and HO-1 were determined by Western blot. Compared with controls, the low and high-dose blueberry serum groups significantly inhibited the HSC proliferation (P < 0.05). It had the same inhibitory effects as the positive control serum group (P > 0.05). ColI of culture supernatant obviously decreased (P < 0.05). And the expression levels of α-SMA in low and high-dose blueberry serum groups decreased significantly (P < 0.05). And there were similar effects in low & high-dose blueberry serum and positive control serum groups. Western blot showed that the expressions of Nrf2 and HO-1 in blueberry and positive control serum groups were higher than that in control group. And the increment was more significant in the low and high-dose blueberry serum groups (P < 0.05). Blueberry can significantly inhibit the proliferation and activation of HSC and reduce the synthesis of extra-cellular matrix. It may have potential preventive and protective effects on hepatic fibrosis. The mechanism may be related to an elevated expression of HO-1 through the Nrf2 pathway.

Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation

Abstract While purified bacterial cells and DNA – the signature of life – from soil and sediment matrices have been extensively studied in a wide range of environments and in different microbial ecosystems, the paucity of data on DNA extraction from contaminated sediments emphasizes the need for further research on the isolation and quantification of bacterial cells and DNA in sediments. Consequently, the Nycondez gradient centrifugation method was applied to extract bacterial cells from contaminated and uncontaminated sediments. Quantitative estimates of recovered bacterial cells were obtained from direct counts performed using DAPI (4′,6′-diamino-2-phenylindole hypochloride) staining couples with fluorescence microscopy and indirect counts (colony-forming units). The estimation was improved by using an efficient method of comparing sediment types composed of quantifying bacterial densities in three steps: S1 the initial freshwater sediments; S2 the first supernatant recovered after mixing the sediments with sodium hexametaphosphate solution followed by centrifugation; and S3 the extracted cells. Total and extracellular DNA were extracted and quantified in each of the three steps. Additional analysis of faecal indicator bacteria (FIB) including E. coli and Enterococcus (ENT) was also performed in each step. The results display considerable variability in the quantity of bacteria cells depending on sediment type, ranging from 1.2 × 105 to 6.2 × 109 cell g−1 dry sediments. The treatment with sodium hexametaphosphate solution (2%) leads to the desorption of bacterial populations which were firmly adsorbed on contaminated sediment surfaces resulting in more than 90% of the FIB being recovered. The Nycondez density gradient centrifugation method makes it possible to extract bacterial cells from freshwater sediments without extracellular DNA so it is ideal for metagenomic analysis of bacteria.

Subproteomic analysis of the mitochondrial proteins in rats 24 h after partial hepatectomy

A 70% partial hepatectomy (70%PHx) induces cell proliferation until the original mass of the liver is restored. Mitochondria are involved directly in the process of liver regeneration (LR); however, their role in the early phase of LR is not clear. In an attempt to identify mitochondrial proteins that are correlated with the early phase of LR, we obtained a mitochondrial fraction via Nycodenz(R) density gradient centrifugation and subcellular proteomic analysis was performed. The mitochondrial proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Compared to the sham-operation control group, 3 proteins were up-regulated and 22 proteins were down-regulated at 24 h after 70%PHx. We identified 22 differentially expressed proteins that were associated with carbohydrate metabolism, lipid metabolism, the respiratory chain and oxidation-phosphorylation, biotransformation and other metabolic pathways. Prohibitin, a proliferation-regulating protein that was down-regulated at 24 h after PHx, was analyzed by applying RNAi (PHBi) with BRL-3A. This demonstrated a decreased mitochondrial membrane potential, implying a potential role in maintaining mitochondrial integrity. These results indicated that hepatic mitochondrial adaptations to LR after 70%PHx affect various cellular metabolic pathways, which advances our knowledge of the role of mitochondria in the early phase of LR.

Recovery and quantification of bacterial cells associated with streambed sediments

Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the 3H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.

Presence and Some Characteristics of Peroxisomes in Immortalized Human Trophoblast Cells

Using morphological and biochemical (Western blot and cell fractionation) methods, we investigated whether peroxisomes are present in human extravillous trophoblast cells. Immortalized extravillous trophoblast cells (TCL-1) were incubated in the presence or absence of 0.5 mM clofibric acid for 3 d. In immunofluorescence staining of trophoblast cells with antibodies anti-catalase and anti-acyl-CoA oxidase (marker enzymes of peroxisomes), electron microscopy and immunoelectron microscopy, peroxisomes were detected in the cells. The size and number of peroxisomes in the trophoblast cells were smaller than those in rat liver. The number of peroxisomes was increased by clofibric acid. In Western blot experiment with antibodies anti-peroxisomal enzymes of beta-oxidation system, densitometric analysis revealed approximately two fold increase in staining by clofibric acid. When we performed cell fractionation experiment using catalase as one of the peroxisomal marker enzymes, the highest activity of catalase was found in the light mitochondrial fraction. Specific activity of catalase in the light mitochondrial fraction was significantly increased to about 1.3 times higher than the control value by clofibric acid treatment. Upon Nycodenz density gradient centrifugation, the catalase activity was concentrated in the density fraction around 1.14-1.15. These findings suggest that microperoxisomes, which have a density smaller than those of rat hepatic peroxisomes, do exist in human extravillous trophoblast cells. It may also be possible to proliferate human peroxisomes in limited quantities using peroxisome proliferator of rodents.

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Cryopreservation of Primordial Germ Cells (PGCs) from White Leghorn Embryos Using Commercial Cryoprotectants

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the large size of their yolk structure. Cryopreservation of primordial germ cells (PGCs) is an alternative way to preserve both female and male genetic material in chicken. In the present study, fertilized eggs were incubated for about 53 hours to obtain embryos at stage 14-16. The blood of the whole embryo was collected from the terminal vein or dorsal aorta using a fine glass micro pipette under a microscope. The PGCs were then purified using Nycodenz density gradient centrifugation. Four commercially available cryoprotectants (A, B, C and D) were used to preserve the PGCs, and DMSO was used as a control. The average recovery rate of PGCs after thawing was 58.2%, 52.9%, 46.5% and 39.4% with the A, B, C and D treatments, respectively. There was no significant difference between the A, B and C treatments and control (DMSO), which showed an average recovery rate of 49.9%. However, the recovery rate obtained using D cryoprotectant was significantly lower than using treatment A. The average viability of the PGCs after thawing were 86.0%, 82.3%, 81.1% and 84.8% for cryoprotectants A, B, C and D, respectively, and the control (DMSO) was 85.3%. There was no statistically significant difference between the four treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos ; however, the use of cryoprotectant D should be avoided since the recovery rate was significantly lower, although the viability was not affected.

Evaluation of quantitative and qualitative recovery of bacterial communities from different soil types by density gradient centrifugation

Extracting and purifying a representative fraction of bacteria from soil is necessary for the application of many techniques of microbial ecology. Here the influence of different soil types on the quantitative and qualitative recovery of bacteria by soil grinding and Nycodenz density gradient centrifugation was investigated. Three soils presenting contrasted physicochemical characteristics were used for this study. For each soil, the total (AODC: acridine orange direct count) and culturable (cfu: colony-forming units) bacterial densities were measured in three distinct fractions: (i) the primary soil, (ii) the soil pellet (soil remaining after centrifugation), and (iii) the extracted cells. The automated–ribosomal intergenic spacer analysis (A-RISA) was used to characterize the community structure directly from the DNA extracted from each fraction. The physicochemical characteristics of soils were found to influence both the efficiency of bacterial cell recovery and the representativeness of the extracted cells in term of community structures between the different fractions. Surprisingly, the most representative extracted cells were obtained from the soil exhibiting the lowest efficiency of cell recovery. Our results demonstrated that quantitative and qualitative cell recovery using Nycodenz density gradient centrifugation are not necessarily related and could be differentially biased according to soil type.

Proteome Analysis of the Rice Etioplast: Metabolic and Regulatory Networks and Novel Protein Functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.

HISTONE H3 MODIFICATIONS IN RAT HEPATIC STELLATE CELLS BY ETHANOL

Hepatic stellate cells (HSCs) play critical roles in the development of hepatic fibrosis caused by various agents including alcohol. Ethanol causes post-translational modification in histone. The goal of this study is to investigate whether ethanol affected acetylation and methylation of histone H3 in rat HSCs. We isolated and separated HSCs using collagenase perfusion of liver followed by Nycodenz density gradient centrifugation. HSCs were divided and treated with different concentrations of ethanol for various times. Histone was isolated using acid extraction method. Acetylation and methylation of histone H3 at Lys9 was analysed by both western blot and fluorescein isothiocyanate (FITC) immunochemical stain. Acetylation of histone H3 at Lys9 (Ac-H3-lys9), Lys14 (Ac-H3-Lys14), Lys18 (Ac-H3-lys18), or Lys23 (Ac-H3-lys23) was checked by western blotting. At lysine 9, ethanol caused dose-dependent increase of Ac-H3 up to 200 mM. Ac-H3-lys9 increased with a maximum of 86-fold at 72 h and 200 mM ethanol treatment, and decreased thereafter. This increase was confirmed by both western blotting and FITC stain. At high dose, ethanol increased acetylation of histone H3 at Lys23 (Ac-H3-lys23), but it had no effect on Ac-H3-lys14 or Ac-H3-lys18. The intensity of the FITC-labelled dimethyl-histone H3 at Lys9 (Me-H3-lys9) antibody appeared to decrease slightly with increasing dose of ethanol. But this did not appear to change when monitored by western blotting. Ethanol caused dose and time-dependent increase in acetylation of histone H3 at Lys9, but not at Lys14 or Lys18. Compared with hepatocytes the Ac-H3-lys9 in HSCs required longer ethanol exposure. Levels of Me-H3-lys9 seemed to remain unaltered. Thus increase in Ac-H3-lys9 represents a nuclear-chromatin modification event in HSCs exposed to ethanol.