Lysis Solution Research Articles

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.

Determination of ascorbic acid in individual rat hepatocyte cells based on capillary electrophoresis with electrochemiluminescence detection

A novel method to detect ascorbic acid (AA) in individual rat hepatocyte cells was developed by combining CE with electrochemiluminescence (ECL) based on tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)(3)2+). A single cell, followed by 0.1% SDS as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. After optimizing the analytical conditions, the RSDs of migration time and peak height were 0.38% and 2.6% for 1.0x10(-5) M AA (n=10), respectively. The linear range of AA was from 1.0x10(-8) to 5.0x10(-5) M with a correlation coefficient of 0.9979 and the LOD (S/N=3) was estimated to be 1.0x10(-8) M. This method has been successfully applied to determine AA in single rat hepatocytes and the amount of AA in seven rat hepatocytes ranged from 16 to 62 fmol. The above results demonstrated that CE coupled with ECL is convenient, sensitive, and will become an attractive alternative method for single-cell analysis.

Development of a fully integrated microfluidic system for sensing infectious viral disease

An active micromixer system utilizing the magnetic force was developed and examined for its ability to facilitate the mixing of more than two fluid flows. The mixing performance of the active micromixer was evaluated in aqueous–aqueous systems including dyes for visual observation. A complete analytical microfluidic system was developed by integrating various functional modules into a single chip, thus allowing cell lysis, sample preparation, purification of intracellular molecules, and subsequent analysis. Upon loading the cell samples and lysis solution into the mixing chamber, the integrated microfluidic device allows efficient cell disruption by rotation of a micromagnetic disk and control of mixing time using the Teflon‐coated hydrophobic film as a microvalve. This inflow is followed by separating the cell debris and contaminated proteins from the cell lysate sample using the acrylamide (AAm)‐functionalized SPE. The inflow of partially purified cell lysate sample containing the gold binding polypeptide (GBP)‐fusion protein was bound onto the gold micropatterns by means of its metal binding affinity. The GBP‐fusion method allows immobilization of proteins in bioactive forms onto the gold surface without surface modification suitable for studying antigen–antibody interaction. It was used for the detection of severe acute respiratory syndrome (SARS), an infectious viral disease, as an example case.

Determination of ascorbic acid in individual rat hepatocyte by capillary electrophoresis with electrochemical detection

A method for the direct determination of ascorbic acid (AA) in individual rat hepatocyte based on capillary electrophoresis (CE) coupled with electrochemical detection (ECD) using a new kind of homemade carbon fiber micro-disk bundle electrode has been described. Individual rat hepatocytes were injected into a fused-silica capillary with an inner diameter of 25 μm, and lysed by 0.1% sodium dodecylsulfate (SDS) as cell lysis solution. The following conditions were suitable for the determination of AA: running buffer, 1.83 × 10 −2 mol/l Na 2HPO 4–1.70 × 10 −3 mol/l NaH 2PO 4 (pH 7.8); separation voltage, 20.0 kV; detection potential, 0.80 V ( vs. saturated calomel electrode (SCE)). The concentration limit of detection (LOD) of the method was1.7 × 10 −6 mol/l at a signal-to-noise ( S/N) ratio of 3, and the mass LOD was 3.0 fmol. The linear dynamic range was from 5.0 × 10 −6 to 5.0 × 10 −4 mol/l with a correlation coefficient of 0.9962 for the injection voltage of 5.0 kV and injection time of 10 s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the peak current. This method was successfully applied to AA determination in rat hepatocyte. The recovery was between 91% and 97%, and the amount of AA in single rat hepatocyte ranged from 28 to 63 fmol.

Control of pH during plasmid preparation by alkaline lysis of Escherichia coli

Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ‘‘ghost bands” of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing.

Selection and Identification of Acute Proteins with Proteomic Techniques in Rhinencephalon of Cyprinus Carpio After Wounded Acutely

A combined available approach of low osmosis and freeze-soluble with liquid nitrogen was used to disrupt the rhinencephalon tissue of Cyprinus carpio (CC). A centrifugal method with low speed was used to extract proteins in rhinencephalon tissue of CC (CCRT), and high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map with 1200 protein spots was obtained. In this study, several techniques were used for extracting and separating proteome in CCRT. Three lysis solutions were prepared to disrupt the tissue in the fish, and used to extract proteome, water-soluble proteins and fat-soluble proteins, respectively. Both water-soluble proteins and fat-soluble protein from the CCRT were effectively separated by an optimized 2D-PAGE method with high resolution. With treating by 10% acetic acid, six acute proteins in CCRT were selected by proteomic techniques, and further identified by peptide mass fingerprinting (PMF) and database research, respectively. Three of these differential proteins can be primarily defined to be 70 kD heat-shock protein, DNA ligase IV, and β 5 tubulin. It was proposed that these proteins may be used as markers to study the repairing pathway and mechanism in brain once wounded.

Cellular volume and marker expression in human peripheral blood apheresis stem cells

Coulter volume is far more accurate measure of cell volume than forward angle light scatter. In this report, we have used Coulter volume to determine the mean cell volume and diameter of normal human peripheral blood cells and hematopoietic progenitor cells obtained by apheresis (HPC-A) from patients with hematological malignancies. Fresh peripheral blood samples (treated with Beckman Coulter IMMUNOPrep erythrocyte lysis solution), HPC-A samples (treated with BD Biosciences FACSLysing solution), or processed by Ficoll Hypaque sedimentation method were stained with CD45-FITC and PE-labeled CD34, CD90, CD117, and CD133 antibodies and analyzed for electronic volume and two color fluorescence. The mean electronic volume and diameter of mononuclear cells from fresh peripheral blood samples prepared with IMMUNOPrep were lymphocytes (191 microm(3), 7.16 microm), monocytes (370 microm(3), 9.91 microm), and granulocytes (328 microm(3), 8.56 microm). In mononuclear cells of HPC-A samples prepared by Histopaque-1077 sedimentation, the lymphocytes had volume and diameter of 311 microm(3), 8.4 microm, monocytes were 486 microm(3), 9.76 microm, and granulocytes were 515 microm(3), 9.95 microm. In contrast, HPC-A samples prepared after lysis with FACSLysing solution had mean electronic volume and diameter of lymphocytes (414 microm(3), 9.25 microm), monocytes (797 microm(3), 11.5 microm), and granulocytes (670 microm(3), 10.85 microm). Cell volume of mononuclear cells in the HPC-A samples prepared by Histopaque-1077 sedimentation method was correlated with the expression of stem cell markers CD34, CD90, CD117, and CD133. CD90 positive cells had the smallest mean electronic volume of 299.93 microm(3) when compared with cells with positive expression of CD133 (322 microm(3)), CD117 (349 microm(3)), CD34 (407 microm(3)), and CD45 (453 microm(3)). Correlation of cell volume with stem cell marker expression may allow for the identification of small stem cells, which may not express the conventional markers used for the identification of stem cells in HPC-A samples.

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlOx membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.

Application of cytogenetic endpoints and comet assay on human lymphocytes treated with atorvastatin in vitro

This study investigated the genotoxic potential of atorvastatin on human lymphocytes using comet assay, structural chromosome aberrations (CA) and sister-chromatid exchange (SCE) analysis. Lymphocyte cultures were treated with a single drug at a concentration of 30.21 ng/mL. For comet assay, cells exposed to atorvastatin for 24 h, 48 h and 72 h were embedded in agarose slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For analysis of structural CA, cells were grown on medium for 48 h and for SCE analysis for 72 h. Structural CA did not induce significant damage to the genome, although a higher CA frequency was observed in cells treated with atorvastatin for 3 h, 20 h and 48 h than in control samples. Results of the SCE analysis did show statistically significant differences in the mean SCE number between atorvastatin-exposed and control human lymphocytes and between different exposure times. Comet assay also showed increased DNA damage caused in atorvastatin-exposed human lymphocytes than in corresponding control cells for exposure times of 24 h, 48 h and 72 h for the tail length and for 72 h for the tail moment. Results obtained in this study point to the significance of biological indicators providing information on the primary genome damage after long-term exposure, which can help to establish drug therapeutic concentrations that do not put patients with high blood cholesterol to a greater treatment-related risk.

Regulation of Human Lung Adenocarcinoma Cell Migration and Invasion by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.

Squalene Synthase, a Determinant of Raft-associated Cholesterol and Modulator of Cancer Cell Proliferation

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.

Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes

Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.

Phosphorylation of PKCdelta participates in the toxicity of 6-hydroxydopamine on dopaminergic neuroblastoma cell

To investigate the role of phosphorylation of protein kinase C (PKC) delta in the toxicity of 6-hydroxydopamine (6-OHDA) to the death of dopaminergic neurons. Human neuroblastoma cells of the line SH-SY5Y were cultured 6-OHDA of the concentrations of 0, 50, 100, 200, and 400 micromol/L was added to observe its toxicity. Rottlerin (PKCdelta inhibitor, 2 micromol/L), bisindolylmaleimide (Bis, general PKC inhibitor, 10 nmol/L), Gö6976 (calcium-dependent PKC inhibitor, 5 nmol/L), and phobol-12-myristate-13-acetate (PMA, PKC activator, 100 nmol/L) were added into the culture fluid of another SH-SY5Y cells respectively, and then (1) culture fluid of equal volume was added for 18 h so as to observe there effects on the survival of the SH-SY5Y cells, or (2) 100 micromol/L 6-OHDA was added to observe the effects of intervention on PKC on the survival of the SH-SY5Y cells by using MTT assay. Cell lysis solution with phosphatase inhibitor was used to lyse the culture cells to extract plasma protein. Western blotting was used to detect the expression of phosphorylated PKCdelta. MTT assay showed that all different concentrations (50 - 400 micromol/L) of 6-OHDA significantly and dose-dependently caused cell death with an EC50 of 92 micromol/L. Pretreatment with rottlerin and Bis alone did not influence the survival of the cells significantly,. However, the survival rate of the cells pretreated by Gö6976 alone was 92.3% +/- 3.2% that of the control group (P < 0.01), and the survival rate of the cells pretreated by PMA was 49.5% +/- 1.0% that of the control group (P < 0.01) Pretreatment of rottlerin decreased the death rate of the cells treated with 6-OHDA to 30.4% +/- 1.6% and conferred significant protection against 6-OHDA neurotoxicity by 57% +/- 6% compared to that of the cells treated by 6-OHDA alone (P < 0.01). However, Bis and Gö6976 did not affect the 6-OHDA-induced cell damage. Pretreatment of PMA increased the death rate of the cells treated with 6-OHDA to 67.1% +/- 2.2% and significantly aggravated 6-OHDA-induced cell toxicity by 66% +/- 9% (P < 0.01). Western blotting showed that 6-OHDA administration increased the expression of phosphorylated PKCdelta, pretreatment with Rottlerin inhibited such increase, PMA promoted such increase, and Bis and Gö6976 did not influence such increase. Inhibition of PKCdelta phosphorylation with rottlerin ameliorates the neurotoxicity evoked by 6-OHDA, and activation of PKCdelta phosphorylation by PMA aggravates neurotoxicity, which implicating that this kinase participates in the 6-OHDA-induced neurotoxicity and Parkinsonian neurodegeneration.

Association of Kv1.5 and Kv1.3 Contributes to the Major Voltage-dependent K+ Channel in Macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

Calcium-mediated Transient Phosphorylation of Tau and Amyloid Precursor Protein Followed by Intraneuronal Amyloid-β Accumulation

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Role of adipose differentiation-related protein in lung surfactant production: a reassessment

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.

Low levels of clustered oxidative DNA damage induced at low and high LET irradiation in mammalian cells

DNA double-strand breaks (DSBs) and locally multiply damaged sites (LMDS) induced by ionizing radiation (IR) are considered to be very genotoxic in mammalian cells. LMDS consist of two or more clustered DNA lesions including oxidative damage locally formed within one or two helical turns by single radiation tracks following local energy deposition. They are thought to be frequently induced by IR but not by normal oxidative metabolism. In mammalian cells, LMDS are detected after specific enzymatic treatments transforming these lesions into additional DSBs that can be revealed by pulsed-field gel electrophoresis (PFGE). Here, we studied radiation-induced DSBs and LMDS in Chinese hamster ovary cells (CHO-K1). After addition of the iron chelator deferoxamine (DFO) or the antioxidant glutathione (GSH) to the cell lysis solution, we observed reduced spontaneous DNA fragmentation and a clear dose-dependent increase of radiation-induced DSBs. LMDS induction, however, was close to background levels, independently of dose, dose rate, temperature and radiation quality (low and high LET). Under these experimental conditions, artefactual oxidative DNA damage during cell lysis could not anymore be confounded with LMDS. We thus show that radiation-induced LMDS composed of oxidized purines or pyrimidines are much less frequent than hitherto reported, and suggest that they may be of minor importance in the radiation response than DSBs. We speculate that complex DSBs with oxidized ends may constitute the main part of radiation-induced clustered lesions. However, this needs further studies.

SIMPLE METHOD FOR RNA PREPARATION FROM CYANOBACTERIA1

A simple and rapid method is presented for the preparation of RNA from various cyanobacteria. Unlike other methods that require a lysis solution, lysozymes, or proteinase K, the proposed method, called the bead–phenol–chloroform (BPC) method, uses silica/zirconia beads, phenol, and chloroform to break the cells and extract RNA more efficiently. Experiments confirm that the BPC method can successfully isolate total RNA from various cyanobacterial strains without DNA contamination, and the extracted RNA samples have a relatively high purity, concentration, and yield. Furthermore, the BPC method is more rapid, simple, and economical when compared with previously reported methods.