Nycodenz Gradient Research Articles

Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update.

In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.

Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells.

To obtain meaningful results of hepatic stellate cell (HSC) function, it is crucial to use highly pure HSC populations. Our aim was to optimize HSC isolation from mice livers without exploiting the characteristically transient vitamin A autofluorescence of HSC. HSCs were isolated from C57BL/6 mice using a two-step collagenase digestion and Nycodenz gradient separation followed by CD11b-negative sorting step in order to remove contaminating macrophages and dendritic cells. Isolated cells were analyzed for yield, viability, purity, and potential new markers using immunofluorescence and flow cytometry. We obtained a yield of 350,595 ± 100,773 HSC per mouse liver and a viability of isolated cells of 92.4 ± 3.1%. We observed a low macrophage/dendritic cell contamination of 1.22 ± 0.54%. Using flow cytometry, we demonstrated that CD38 was expressed at the surface of HSC subpopulations and that all expressed intracellular markers specific for HSC in the liver. This isolation method, avoiding fluorescent activated cell sorting (FACS), allowed isolation of HSCs with high purity. Further, flow cytometry analysis suggests that CD38 may be a reliable marker of HSCs and may include subpopulations of HSCs without retinoid droplets.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

A Scalable Purification Method for Mitochondria from Trypanosoma brucei.

Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100mL or as large as 24L.

High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles

BackgroundHepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful.MethodsIn a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA.ResultsThe HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes.ConclusionsThe results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.

EVALUATION OF RAT LEYDIG CELL CULTURE COLLECTED WITH NYCODENZ GRADIENT IN PRODUCING TESTOSTERONE IN VITRO

The aim of this study was to evaluate the ability of rat’s Leydig cells collected with Nycodenz gradient in producing testosterone in vitro. Leydig cells were collected using 5 column of Nycodenz gradient (4, 8, 10, 12, and 15%) and cells were evaluated regarding its concentration, viability, and purity of Leydig cells. Media used to cultured Leydig cells were Dulbecco’s Modified Eagle’s Medium (DMEM)+10% newborn calf serum (NBCS); DMEM+10% NBCS+2,5 IU/mL human chorionic gonadotrophin (hCG); DMEM+10% NBCS+ 5 µg/mL insulin, 10 µg/mL transferrin, and 5 µg/mL Se (ITS); DMEM+10% NBCS+hCG+ITS at 5% CO 2 incubator with temperature of 37.5° C for 3 days. Culture medium was collected every day for testosterone analysis with enzyme-linked immunosorbent assay (ELISA). By adding ITS to the medium, Leydig cells concentration was significantly increased (8.92x10 6 cells/mL) compared to medium with serum (7.74x10 6 cells/mL) or hCG (7.68x10 6 cells/mL) (P<0.05). ITS and hCG in medium significantly increased Leydig cells concentration (10.40x10 6 cells/mL) at day 3 of culture (P<0.05). The result of parallelism test showed that the assay obtained good validity to measure testosterone concentration in culture medium. Testosterone in medium was detected at 1.80-2.60 ng/mL at day 1 of culture. In conclusion, Leydig cells collected with Nycodenz gradient had no effect to testosterone secretion from Leydig cells in vitro.

Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes.

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.

Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro

During hepatitis B virus (HBV) infections subviral particles (SVP) consisting mainly of hepatitis B surface antigen are present at much higher concentration than viral particles (VP) in serum. To investigate reasons for this excess of SVP production, SVP and VP were fractionated on a Nycodenz gradient and analyzed for HBV infection of HepG2-NTCP cells with and without anti-HBs antibodies. Our findings showed that SVP significantly reduced the neutralization of VP by anti-HBs, while SVP had little effect on viral entry, supporting the assumption that SVP serve as decoy facilitating cell-to-cell spread of HBV in the presence of neutralizing antibodies.

Isolation of Peroxisomes from Mouse Brain Using a Continuous Nycodenz Gradient: A Comparison to the Isolation of Liver and Kidney Peroxisomes.

In the central nervous system (CNS) peroxisomes are present in all cell types, namely neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Brain peroxisomes are smaller in size compared to peroxisomes from other tissues and are therefore referred to as microperoxisomes. We have established a purification procedure to isolate highly purified peroxisomes from the central nervous system that are well separated from the endoplasmic reticulum and mitochondria and are free of myelin contamination. The major difficulty in purification of brain peroxisomes compared to peroxisomes from liver or kidney is the presence of large amounts of myelin in the CNS, which results in contamination of the subcellular fractions. Hence, the crucial step of the isolation procedure is the elimination of myelin by the use of a sucrose gradient, since without the elimination of myelin no significant enrichment of purified peroxisomes can be achieved. Another difficulty is that in brain tissue the abundance of peroxisomes decreases significantly during postnatal development. We provide a detailed protocol for the isolation of peroxisomes from mouse central nervous system as well as a protocol for the isolation of peroxisomes from the liver and kidney using a continuous Nycodenz gradient.

42 Effect of Immunosuppressive Therapies on Mucinous Pancreatic Cyst Progression

AIM: A remodelling of the pancreatic cancer tumour microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. We investigated whether the aspartic protease cathepsin D contributes to proliferation, migration and fibrogenesis of pancreatic cancer cells and its expression affects the survival of pancreatic cancer patients. MATERIALS & METHODS: Mouse pancreatic stellate cells (PSC) were isolated by Nycodenz gradient. PSC and murine pancreatic cancer PD6606 cells were used for Western blotting and immunofluorescence staining. Cathepsin D activity was inhibited with pepstatin and its expression silenced with siRNA. TGFβ1 was measured using microplate sandwich ELISA and proliferation by standard MTT assay. Tumour tissue microarrays of 404 patients from the ESPAC-3 trial (all after PDAC-resection) were stained with anti-cathepsin D (C-20) antibody and patients with the highest quartile H-score (cut-off 22.35) were compared with those in the lowest quartile using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS: Inhibition of cathepsin D in ductal adenocarcinoma cells resulted in significantly reduced proliferation and migration. Cathepsin D expression increased in parallel with PSC trans-differentiation to myofibroblasts. Silencing (siRNA) or inhibiting cathepsin D decreased PSC proliferation by 20-30%. There was a time-dependent activation of latent TGF β1 and cathepsin D cleaved latent TGF β1, leading to its activation. Inhibition of cathepsin D decreased activated TGF β1. In 1824 tissue microarray cores from 382 patients (94.6%) median overall patients' survival was 23.85 months (95% CI 21.0-26.2). Median survival for patients in the highest quartile cathepsin D H-score was 21.2 months (95% CI 17.3-24.8) and in the lowest quartile 27.2 months (95% CI 24.1-31.2) χLR,1DF = 4.33; P = 0.0374. DISCUSSION: In pancreatic cancer, cathepsin D expression not only regulates fibrogenesis and stromal development but also ductal adenocarcinoma cell proliferation and migration. These mechanisms make cathepsin D a driver of pancreatic cancer growth and account for the poor prognosis of patients with high cathepsin D expression in PDAC.

43 Long-Term Outcomes of IPMNs With Consensus Guidelines Criteria for Resection Undergoing Nonoperative Management

AIM: A remodelling of the pancreatic cancer tumour microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. We investigated whether the aspartic protease cathepsin D contributes to proliferation, migration and fibrogenesis of pancreatic cancer cells and its expression affects the survival of pancreatic cancer patients. MATERIALS & METHODS: Mouse pancreatic stellate cells (PSC) were isolated by Nycodenz gradient. PSC and murine pancreatic cancer PD6606 cells were used for Western blotting and immunofluorescence staining. Cathepsin D activity was inhibited with pepstatin and its expression silenced with siRNA. TGFβ1 was measured using microplate sandwich ELISA and proliferation by standard MTT assay. Tumour tissue microarrays of 404 patients from the ESPAC-3 trial (all after PDAC-resection) were stained with anti-cathepsin D (C-20) antibody and patients with the highest quartile H-score (cut-off 22.35) were compared with those in the lowest quartile using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS: Inhibition of cathepsin D in ductal adenocarcinoma cells resulted in significantly reduced proliferation and migration. Cathepsin D expression increased in parallel with PSC trans-differentiation to myofibroblasts. Silencing (siRNA) or inhibiting cathepsin D decreased PSC proliferation by 20-30%. There was a time-dependent activation of latent TGF β1 and cathepsin D cleaved latent TGF β1, leading to its activation. Inhibition of cathepsin D decreased activated TGF β1. In 1824 tissue microarray cores from 382 patients (94.6%) median overall patients' survival was 23.85 months (95% CI 21.0-26.2). Median survival for patients in the highest quartile cathepsin D H-score was 21.2 months (95% CI 17.3-24.8) and in the lowest quartile 27.2 months (95% CI 24.1-31.2) χLR,1DF = 4.33; P = 0.0374. DISCUSSION: In pancreatic cancer, cathepsin D expression not only regulates fibrogenesis and stromal development but also ductal adenocarcinoma cell proliferation and migration. These mechanisms make cathepsin D a driver of pancreatic cancer growth and account for the poor prognosis of patients with high cathepsin D expression in PDAC.

Definition of an HBsAg to DNA international unit conversion factor by enrichment of circulating hepatitis B virus forms.

Hepatitis B (HBV) virus infection is characterized by the overproduction of subviral particles (SVP) over infectious Dane particles (VP). Precise regulation of the ratio between these forms is unknown, but its fluctuation may have a clinical impact. An enrichment method was applied to assess the SVP/VP ratio in chronically infected patients (CHB) and to compare the sensitivity of HBs antigen (HBsAg) and DNA detection methods. Plasmas from 9 genotype A-D CHB patients were fractionated on Nycodenz(®) gradients, and both HBV DNA and HBsAg were quantified in each collected fraction using standardized techniques expressed in IU/mL. Infection of primary human hepatocytes (PHHs) was performed with crude or fractionated plasma. Independently of the genotype, all plasmas showed a similar rate-zonal separation profile characterized by a bottom DNA-enriched peak surmounted by HBsAg-enriched fractions. Inoculation of PHH with plasma-derived VP-enriched fractions led to long-lasting production of virus in cell supernatants with a SVP/VP ratio similar to that observed in patient plasmas. In the VP fraction, one IU of HBsAg corresponded to approximately 5 million IU of HBV DNA. Rate-zonal gradient separation directly applied on patient plasma allows a better insight into the distribution of VP in HBeAg-positive CHB carriers. This study highlights the sensitivity difference of the techniques classically used to monitor HBV infection and indicates that VP-associated HBsAg contributes modestly to the overall amount of total circulating HBsAg in CHB. Such a fractionation approach should help to understand the fine regulation of HBsAg production over replication at different stages of CHB.

Visfatin: an adipokine activator of rat hepatic stellate cells.

The present study was conducted to investigate the effects of visfatin on the activation of hepatic stellate cells (HSC) and the possible underlying mechanism. HSC were isolated from the livers of Sprague‑Dawley rats by insitu perfusion of collagenase and pronase and a single‑step density Nycodenz gradient. The culture‑activated cells were serum‑starved and incubated with different concentrations of recombinant visfatin (0, 25, 50, 100 or 200ng/ml) for 24h. The expression of α‑smooth muscle actin (α‑SMA), collagen typesI and III and connective tissue growth factor (CTGF) were then measured by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. The results demonstrated that 100 and 200ng/ml concentrations of visfatin induced the expression of α‑SMA in culture‑activated rat HSC, which was accompanied by a significant increase in collagen typesI and III, as confirmed by western blot and RT‑qPCR analyses. In addition, treatment of the HSC with certain concentrations of visfatin upregulated the expression of CTGF. These findings suggested that visfatin activated HSC and induced the production of collagen typesI and III.

Development of &amp;lt;i&amp;gt;in vitro&amp;lt;/i&amp;gt; culture of rat Leydig cells after purification with Nycodenz gradient

The study examined the effect of human Chorionic Gonadotrophin (hCG) and/or Insulin Transferring Sodium Selenite (ITS) on the proliferation and development of rat Leydig cells purified by Nycodenz gradient. Leydig cells purity, viability and proliferation after purification and 3 days of cultured were evaluated. Leydig cells 1 × 106 cells/ml were cultured in DMEM containing 10% Newborn Calf Serum (NBCS) and divided into four kinds of treatments 1) as a control, 2) control + 2.5 IU/ml hCG, 3) control + ITS (5 μg/ml insulin, 10 μg/ml transferrin, 5 μg/ml Se), and 4) control + hCG + ITS. Leydig cells purification results showed 91.40% of purity, viability was 98.17% and concentration 7.03 × 106 cells/ml. The addition of ITS and hCG + ITS in DMEM produced Leydig cell proliferation by 88.35% and 90.64% higher than in controls (86.82%) (p p p

Differentially expressed genes identified by microarray analysis following oxymatrine treatment of hepatic stellate cell

AIM: To investigate the effects of oxymatrine on the gene expression profile of hepatic stellate cell (HSC) and provide novel insights into the mechanism of oxymatrine against hepatic fibrosis. Methods: HSC was isolated from normal SD by in situ perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. MTT colorimetry was used to study the effect of oxymatrine on the proliferation of HSC. Total RNA and mRNA of quiescent HSC, culture-activated HSC and oxymatrine treated HSC were extracted. Effect of oxymatrine on HSC gene expression profile was detected by oligonucleotide microarray analysis with Affymetrix gene chip rat U230A. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Results: Oxymatrine could inhibit the proliferation of HSC in a dose-dependent manner. A total of 4641 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, among which 2702 genes were upregulated, and 1939 genes were down-regulated in activated HSC. cDNA microarray uncovered downregulation of 56 genes in response to oxymatrine, the representative genes including alpha 2 type I procollagen, alpha-1 type I collagen, tissue inhibitor of metalloproteinase 1, interleukin 1 beta, early growth response 1, chemokine ligand 2, chemokine ligand 1, CTGF, TGFβ1. The most enriched GO terms included response to wounding, inflammatory response, cell migration, cell motility, wound healing, TGFβ receptor signaling pathway. KEGG pathway analysis revealed that oxymatrine affected the ECM-receptor interaction, focal adhesion, cytokine-cytokine recaptor interaction, TGFβ signaling pathway, MAPK signaling pathway. There were 37 genes upregulated significantly following oxymatrine treatment. The most enriched GO terms included oxidation reduction, negative regulation of lipoprotein oxidation, regulation of lipoprotein oxidation, steroid metabolic process, regulation of lipase activity. Six genes were confirmed with QPCR, consistent with microarray. Conclusion: The mechanism of oxymatrine in inhibiting liver fibrogenesis is associated with multi-genes and multi-pathways regulation.

Multiple Pathways for High Voltage‐Activated Ca2+ Influx in Anterior Pituitary Lactotrophs and Somatotrophs

The present study demonstrates that a significant proportion of high voltage-activated (HVA) Ca(2+) influx in native rat anterior pituitary cells is carried through non-L-type Ca(2+) channels. Using whole-cell patch-clamp recordings and specific Ca(2+) channel toxin blockers, we show that approximately 35% of the HVA Ca(2+) influx in somatotrophs and lactotrophs is carried through Ca(v) 2.1, Ca(v) 2.2 and Ca(v) 2.3 channels, and that somatotrophs and lactotrophs share similar proportions of these non-L-type Ca(2+) channels. Furthermore, experiments on mixed populations of native anterior pituitary cells revealed that the fraction of HVA Ca(2+) influx carried through these non-L-type Ca(2+) channels might even be higher (approximately 46%), suggesting that non-L-type channels exist in the majority of native anterior pituitary cells. Using western blotting, immunoblots for α(1C) , α(1D) , α(1A) , α(1B) and α(1E) Ca(2+) channel subunits were identified in native rat anterior pituitary cells. Additionally, using reverse transcriptase-polymerase chain reaction, cDNA transcripts for α(1C) , α(1D) , α(1A) and α(1B) Ca(2+) channel subunits were identified. Transcripts for α(1E) were nonspecific and transcripts for α(1S) were not detected at all (control). Taken together, these results clearly demonstrate the existence of multiple HVA Ca(2+) channels in the membrane of rat native anterior pituitary cells. Whether these channels are segregated among different membrane compartments was investigated further in flotation assays, demonstrating that Ca(v) 2.1, Ca(v) 1.2 and caveolin-1 were mostly localised in light fractions of Nycodenz gradients (i.e. in lipid raft domains). Ca(v) 1.3 channels were distributed among both light and heavy fractions of the gradients (i.e. among raft and nonraft domains), whereas Ca(v) 2.2 and Ca(v) 2.3 channels were distributed mostly among nonraft domains. In summary, in the present study, we demonstrate multiple pathways for HVA Ca(2+) influx through L-type and non-L-type Ca(2+) channels in the membrane of native anterior pituitary cells. The compartmentalisation of these channels among raft and nonraft membrane domains might be essential for their proper regulation by separate receptors and signalling pathways.

Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions. HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl(4))-induced fibrosis rat model was also analyzed with Western blotting. Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of type I collagen and transforming growth factor-β1. Wnt5a was upregulated in the fibrotic liver of a CCl(4)-induced fibrosis rat model. Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.

Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria

Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM plants. In this study, we investigated kanamycin-resistant (KmR) and neomycin-resistant (NmR) soil bacterial populations in a 3-year field trial using a commercial GM corn (Zea mays L.) carrying the nptII gene and its near isogenic line. The results showed that a portion (2.3 – 15.6 %) of cultivable soil bacteria was naturally resistant to kanamycin or neomycin. However, no significant difference in the population level of KmR or NmR soil bacteria was observed between the GM and non-GM corn fields. The nptII gene was not detected in any of the total 3000 KmR or NmR isolates screened by PCR. Further, total soil bacterial cells were collected through Nycodenz gradient centrifugation and bacterial community DNA was subjected to PCR. Detection limit was about 500 cells per gram of fresh soil. Our study suggests that the nptII gene was relatively rare in the soil bacterial populations and there was no evidence of gene transfer from a GM corn plant to soil bacteria based on the data from total soil bacterial communities.

Isolation and phenotyping of normal mouse liver dendritic cells by an improved method.

Dendritic cells (DCs) are bone marrow-derived cells, which migrate to lymphoid and non-lymphoid organs via blood. Liver DCs are believed to play an important role in the regulation of hepatic allograft acceptance. However, because of inherent difficulties in isolating adequate numbers of DCs from liver, limited information is available on the phenotype and functions of liver DCs. To address this issue, we isolated DCs from normal C57BL/6 mouse liver using a modified procedure and described their immunophenotypic characteristics. Non-parenchymal cells (NPCs) were obtained by collagenase digestion of perfused liver fragments and density gradient centrifugation (14.5% nycodenz column). After overnight (18 hr) incubation of the NPCs, enrichment for transiently adherent, low- density cells on 13% nycodenz gradients permitted the recovery of low numbers of cells (approximately 1.2-1.5 x 10(5) per liver), many of which displayed distinct DCs morphology (abundant cytoplasm with prominent projections and irregularly shaped nuclei). Flowcytometric analysis revealed that most of these cells were recognized by anti-CD11c (60-70%). The results obtained from double staining with PE and FITC conjugated monoclonal antibodies indicated that these cells were CD11c(+)/MHC-II(+) (53%), CD11c(+)/CD86(+) (53.5%), CD11c(+)/ CD8α(+) (36%) and CD11c(+)/CD11b(+) (45%). These findings indicate that the purity of DCs isolated by nycodenz gradient is higher than other reported methods. Considering the similar ratio of lymphoid (CD11c(+)/CD8α(+)) and myeloid (CD11c(+)/CD11b(+)) DCs in the liver, and the known role of lymphoid DCs in tolerance induction, it seems that this subpopulation of DCs is not the main reason of liver tolerogenecity. Therefore, other factors such as the immaturity of liver DCs or the effect of liver microenvironment on these cells, etc. may explain the acceptance of hepatic allograft.

Role of thiamine pyrophosphate in oligomerisation, functioning and import of peroxisomal 2-hydroxyacyl-CoA lyase

During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His 6-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.