Sodium EDTA Research Articles

Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

The Niemann-Pick C1 Like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homo-logy to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of human NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH2-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. human NPC1L1 contains seven small cytoplasmic loops--four small and three large luminal loops--one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, NPC1, and SCAP. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of the different domains of NPC1L1.

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

Comparative assessment of sodium edta and heparin as anticoagulants for the evaluation of haematological parameters in cultured and feral african catfish (Clarias gariepinus)

This study investigates the effect of anticoagulants on haematological parameters in thirty (30) feral (wild) and thirty (30) pond cultured African catfish (Clarias gariepinus). The anticoagulants used in this study were lithium heparin and sodium ethylenediaminetetraacetic acid (EDTA). Red blood cell (RBC) count, packed cell volume (PCV), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cell (WBC) count and differential count were determined. No significant differences (p >0.05) were observed in the total RBC, WBC and MCHC values in the feral and pond cultured fish. In the pond cultured African catfish, the neutrophil values were significantly higher (p =0.001) than those of the feral African catfish irrespective of the anticoagulant used. The values for PCV, Hb, lymphocytes, eosinophils, MCV and MCHC of the feral African catfish were significantly higher (p <0.05) than those of the pond cultured African catfish irrespective of the anticoagulant used. The monocyte value for the feral fish was significantly higher (p =0.041) when EDTA was used as the anticoagulant. This study therefore concludes that both anticoagulants could be used for haematological evaluation of fish blood. Also, the habitat and environmental conditions should be taken into consideration when analyzing blood samples in fish.

Purification of an infection-related acidic peroxidase from pearl millet seedlings

Higher basal level of peroxidase activity was observed in highly resistant pearl millet cultivar IP 18292. Upon inoculation with downy mildew pathogen, Sclerospora graminicola, up to 60% increase in peroxidase activity was observed in highly resistant seedlings over the period of time. Iso-electric focusing analysis revealed that, two acidic isozymes of peroxidase with the pI of 5.9 and 5.1 present only in IP 18292 pearl millet seedlings. Upon inoculation with downy mildew pathogen, accumulation of these to isozymes was increased. These results indicated the possible involvement of acidic peroxidase in pearl millet defense. To study the nature of the acidic peroxidase which increases upon inoculation was purified from seedlings of highly resistant pearl millet cultivar using DEAE–Sepharose and Sephadex G-100 columns. The purified enzyme has a molecular weight of 21.8 kDa on SDS–PAGE and has a pI of 5.1. The optimum pH for maximum peroxidase activity was found to be at pH 7.0 and was resistant to high temperature (27–60 °C). The K m for H 2O 2 and V max of the enzyme reaction were 5.26 mM and 322.58 units, respectively. Purified peroxidase enzyme was found to be CaCl 2 dependent and both MgCl 2 and ZnCl 2 showed inhibitory effect on enzyme activity. Sodium azide and EDTA inhibited the enzyme and EGTA found to be specific inhibitor of peroxidase.

Enterococcus faecalis Biofilms Eradication by Root Canal Irrigants

The aim of this study was to evaluate the minimal biofilm eradication concentration (MBEC) of sodium hypochlorite (NaOCl), chlorhexidine (CHX), EDTA, and citric and phosphoric acids after 1, 5, and 10 minutes of exposure to biofilms of Enterococcus faecalis. The biofilms grew in the MBEC high-throughput device for 24 hours at 37°C and were exposed to 10 serial two-fold dilutions of each irrigating solution. The viable cell counts were log 10 transformed, and a concentration of an irrigant was considered to eradicate the biofilms when it produced a reduction of ≥ 5 logarithmic units. NaOCl was the most effective agent, capable of eradicating the biofilms after 1 minute at a concentration of 0.00625%. CHX eradicated biofilm after 5 minutes at 2%. EDTA and citric and phosphoric acid solutions were not effective against the biofilms at any concentration or time tested.

Multiresidue determination of tetracyclines, sulphonamides and chloramphenicol in bovine milk using HPLC-DAD

Abstract A high-performance liquid chromatographic method for the simultaneous determination of tetracycline, oxytetracycline, chlortetracycline, sulfamethazine, sulfaquinoxaline, sulfamethoxazole and chloramphenicol in milk has been developed. The determination of these antimicrobials was carried out using HPLC-DAD with a C 18 hybrid column and gradient elution with a mobile phase composed of an aqueous phase of 0.075 mol L −1 sodium acetate, 0.035 mol L −1 calcium chloride and 0.025 mol L −1 sodium EDTA, pH 7 and an organic phase of methanol:acetonitrile, 75:25 v/v. Sample preparation involved protein precipitation followed by solid-phase extraction using a polymeric cartridge. The method was validated and applied for the analysis of pasteurised milk samples commercialised in Brazil. The limits of quantitation for all antimicrobials, with the exception of choramphenicol, were below the maximum residue limit, which indicates that the method is appropriate for the determination of these antimicrobials in milk.

Enhancing process kinetics for mineral carbon sequestration

Abstract The current low-cost process for mineral carbonation involves the direct carbonation of a slurry of magnesium or calcium silicate mineral with supercritical CO 2 . The process is currently limited by the slow reaction kinetics of the carbonation reactions, and in particular the slow dissolution rates of the silicates in weakly acidic conditions. Enhancing the dissolution rate in weakly acidic conditions has been identified as one of the main opportunities for lowering the costs of a direct mineral carbonation process. Serpentine has been identified by its reactivity and abundance as a potential mineral for use in a mineral carbonation process. In this paper we discuss the results of dissolution experiments in which ground serpentine was reacted in weakly acidic aqueous systems containing NH4Cl, NaCl,, sodium citrate, sodium EDTA, sodium oxalate, and sodium acetate. All experiments are carried out at 120 ∘ C and under 20 bars of CO 2 in a batch autoclave. It was found that the sodium salts of citrate, oxalate, and EDTA significantly enhance the dissolution of serpentine under weakly acidic conditions.

Phytate as a novel phosphorus-specific paleo-indicator in aquatic sediments

A reliable geochemical paleo-indicator for phosphorus remains elusive, despite the importance of understanding historical changes in the nutrient status of aquatic ecosystems. We assessed the potential of phytate (salts of myo-inositol hexakisphosphate) as a novel phosphorus-specific paleo-indicator by measuring its concentrations in dated sediments from an embayment in Helsinki, Finland, with a known 200-year history of trophic changes. Phytate was extracted in a solution containing sodium hydroxide and EDTA and detected by solution 31P NMR spectroscopy with spectral deconvolution. Concentrations varied markedly with sediment depth and paralleled previously determined changes in diatom assemblages and geochemical indicators linked to trophic status. In contrast, total sediment phosphorus did not reflect phosphorus inputs to the embayment, presumably due to the mobilization of inorganic phosphate under anoxic conditions during periods of high pollutant loading. Importantly, phytate appeared to be stable in these brackish sediments, in contrast to other organic and inorganic phosphates which declined abruptly with depth. We therefore conclude that phytate represents a potentially important indicator of historical changes in phosphorus inputs to water bodies, although additional studies are required to confirm its stability under conditions likely to be encountered in lakes and coastal ecosystems.

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

A Combination of HNF-4 and Foxo1 Is Required for Reciprocal Transcriptional Regulation of Glucokinase and Glucose-6-phosphatase Genes in Response to Fasting and Feeding

Glucokinase (GK) and glucose-6-phosphatase (G6Pase) regulate rate-limiting reactions in the physiologically opposed metabolic cascades, glycolysis and gluconeogenesis, respectively. Expression of these genes is conversely regulated in the liver in response to fasting and feeding. We explored the mechanism of transcriptional regulation of these genes by nutritional condition and found that reciprocal function of HNF-4 and Foxo1 plays an important role in this process. In the GK gene regulation, Foxo1 represses HNF-4-potentiated transcription of the gene, whereas it synergizes with HNF-4 in activating the G6Pase gene transcription. These opposite actions of Foxo1 concomitantly take place in the cells under no insulin stimulus, and such gene-specific action was promoter context-dependent. Interestingly, HNF-4-binding elements (HBEs) in the GK and G6Pase promoters were required both for the insulin-stimulated GK gene activation and insulin-mediated G6Pase gene repression. Indeed, mouse in vivo imaging showed that mutating the HBEs in the GK and G6Pase promoters significantly impaired their reactivity to the nutritional states, even in the presence of intact Foxo1-binding sites (insulin response sequences). Thus, in the physiological response of the GK and G6Pase genes to fasting/feeding conditions, Foxo1 distinctly decodes the promoter context of these genes and differently modulates the function of HBE, which then leads to opposite outcomes of gene transcription.

Method for the Determination of Lycopene in Supplements and Raw Material by Reversed-Phase Liquid Chromatography: Single-Laboratory Validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.

A Scanning Electron Microscopic Evaluation of the Effectiveness of the F-file versus Ultrasonic Activation of a K-file to Remove Smear Layer

The objective of this study was to compare the effectiveness of F-files and ultrasonics to remove the smear layer from instrumented root canals when irrigated with sodium hypochlorite and EDTA. Sixty healthy human premolar teeth were instrumented with ProTaper file series to F3, and the canals were enlarged with Profiles 35/.06, 40/.06, and 45/.06. The canals were then instrumented with either the F-file or an ultrasonically activated #20 K-file with or without EDTA. The removal of smear layer was visualized using blind scanning electron microscopic micrographs. There appeared to be little difference between the F-file and the ultrasonically activated #20 K-file in removal of the smear layer with or without EDTA. The effect of ultrasonic activation appeared to be self-limiting with high-volume flushes of irrigant. It appears the F-file was not any more beneficial in removing smear layer. Conversely, smear layer removal appears to be mostly influenced by the introduction of an EDTA rinse.

Evaluation of EDTA hematology tubes for collection of blood samples for tests of secondary hemostasis in dogs

To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs. 108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis). Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated. Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples. Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.

Amphoteric Surfactants for PAH and Lead Polluted-Soil Treatment Using Flotation

Polluted soils are a problem of major concern impacting the health of the global environment and human beings. Without any safe technology for polluted soil treatment, most of the contaminated soils go to landfill especially when metals are the pollutants. This research compares the performance of non ionic (Brij 35 and Tween 80) and amphoteric (cocamydopropyl hydroxysultaine (CAS) and coamydopropylbetaine (BW)) surfactants for polycyclic aromatic hydrocarbons (PAHs) and metals (mainly Pb) removal from three contaminated soils. Best removal yields have been obtained using 0.5% (w/w) of CAS surfactant with 46 ± 2% for PAHs and 21 ± 5% for Pb simultaneously removed. Sodium chloride and EDTA have been shown to considerably enhance Pb solubilization (respectively 55 ± 5% and 35 ± 12% for [NaCl] = 5.5 M and [EDTA] = 0.025 M). Flotation technology has been tested as a separating technique of PAHs micelles and Pb from aqueous solution and has given good results for both contaminants (54 ± 7% for both PAH and Pb, using [CAS] = 0.5%, [NaCl] = 5.5 M, pH = 3). Finally, this study proposes a soil washing process using flotation to treat soil polluted with both organic and inorganic compounds. The suggested process concentrates the organic compounds in the froth and the inorganic pollutants in the liquid fraction.

Involvement of Protective Autophagy in TRAIL Resistance of Apoptosis-defective Tumor Cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.

Blood Microvesicles (or Microparticles) Isolation, Identification and Characterization: impact of anticoagulants

Heterogeneous, phospholipid‐rich sub‐micron vesicles (“microvesicles”) shed by activated vascular and blood cells may reflect or influence the coagulant propensity of blood. To determine how the anticoagulant used to collect the blood affects microvesicle (Mv) recovery and characteristics, blood was collected from laboratory volunteers and participants of the Kronos Early Estrogen Prevention Study (KEEPS) into tubes containing either sodium citrate, EDTA or hirudin plus tick anticoagulant peptide (H&amp;T). Microvesicles prepared by double centrifugation were analyzed by flow cytometry to define size and surface markers. There were 2–3 times the number of platelet‐derived (CD61 positive) Mv in preparations from citrate and H&amp;T plasma, relative to numbers in EDTA plasmas. Procoagulant activity of Mv was determined by surface phosphatidylserine (PS) expression using annexin‐V‐fluorescein. Annexin‐V (PS) positive Mv did not differ, but total (PS positive and negative) Mv was 2 fold lower from EDTA plasma compared to citrate and H&amp;T. These results suggest that anticoagulation of blood itself alters microvesicle characteristics. Therefore, standardization and specification of the anticoagulant is warranted to identify potential contribution of microvesicles to cardiovascular pathophysiology. Funded by, NIH HL78638, Kronos Research Institute and Mayo Foundation.

Small outer-membrane lipoprotein, SmpA, is regulated by σ E and has a role in cell envelope integrity and virulence of Salmonella enterica serovar Typhimurium

SmpA is a small outer-membrane lipoprotein that is a component of the essential YaeT outer-membrane protein assembly complex. In Salmonella enterica serovar Typhimurium (S. Typhimurium), expression of the smpA gene was shown to be directed by two promoters, smpAp1 and smpAp2. The more distal promoter, smpAp1, is dependent upon the extracytoplasmic stress response sigma factor sigma(E). An smpA null mutant was constructed in S. Typhimurium SL1344 and was shown to be more sensitive than its wild-type parent to growth at high temperature and in the presence of sodium cholate, SDS plus EDTA, and the hydrophobic antibiotic rifampicin. The lack of SmpA in S. Typhimurium elicits a sigma(E)-dependent stress response. These findings are indicative of altered outer-membrane integrity in the smpA mutant, probably due to a defect in outer-membrane protein biogenesis. SmpA was not important for entry or survival within murine macrophages; however, the S. Typhimurium smpA mutant was attenuated in mice by both the oral and parenteral routes of infection, and SmpA appeared to be most important for the growth of S. Typhimurium at systemic sites.

A Novel Whole Blood Real-Time Microparticles Detection Assay Using Lactadherin-Coupled Dynabeads.

INTRODUCTION: The size and heterogeneity of microparticles (MP) make identification, isolation, and characterization a constant challenge. The current literature reveals a wide assortment of identification and isolation techniques, often resulting in inconsistent or conflicting data. Flow cytometry, while a suitable technique for characterizing MP, suffers from several limitations. Purifying MP requires centrifugation, thereby inducing microvesiculation through shear stress. The constraints of cytometer lasers limit detection of MP size. Annexin V has long been considered the gold standard for identifying phosphatidylserine(PS)-positive MP. However, it only binds PS efficiently in the presence of Ca2+, which may promote microvesiculation as well as aggregation of MP and other soluble proteins. Recent data suggest that lactadherin, a milk fat glycoprotein, binds PS independent of Ca2+ and with greater specificity. We developed a Dynal bead-based assay, which eliminates sample processing while increasing specificity in identifying PS+ and tissue factor-bearing (TF+) MP.METHODS: Lactadherin, isolated from bovine whole milk, were coupled to 4.5 μm Dynabeads. Lactadherin-coated beads (LB, 4 x105 beads/sample) were incubated with 1 mL freshly drawn, citrated whole blood from healthy adult volunteers (N=12) for 10 min at 25°C. LB were collected using a magnetic cell sorter, washed, and labeled with either a cell-specific PE-labeled antibody or FITC-labeled antibodies against TF. Polyclonal anti-TF antibody-coupled Dynabeads were also used to capture TF+ MP, whose origins were then identified by cellular markers. To verify system response, blood samples were stimulated with lipopolysaccharide (LPS) to produce monocytic TF+ MP. Antibody binding was detected and analyzed using an EPICS XL flow cytometer with Expo32 software.RESULTS: LB-bound MP (9% of LB) stained for key cellular markers. Monocyte-derived MP (CD11b) accounted for 12%, whereas platelet (CD41a), endothelial (CD144), and erythrocyte (CD235a)-derived MP accounted for 13%, 52%, and 22%, respectively, verifying that MP arise from 4 major cell types. When circulating levels of various cell types are considered, CD11b+ MP contribute the greatest proportion. LB-bound TF+ MP detected as mean fluorescence intensity (MFI) increased significantly after LPS stimulation (1 μg/mL for 6 h at 37°C) of whole blood (3.2 vs 21.6, p<0.05, Mann-Whitney). CD11b expression was also enhanced (4.9 vs 81.7, p<0.05).CONCLUSIONS: We have developed a novel assay for identifying plasma MP and their cellular origins. This new method offers several advantages over current protocols. Using freshly drawn blood eliminates sample manipulation, shear stress-induced cellular activation, and further microvesiculation. It reduces preparation time, permitting multiple sample analysis in a timely manner. Lactadherin capture of PS+MP allows for sample collection using sodium citrate or EDTA, while reducing calcium-induced non-specific binding. Magnetic sorting of LB removes soluble plasma proteins, thus reducing background noise on flow cytometry analysis. The size of the LB improves the flow cytometer's ability to distinguish signal from noise - greatly improving specificity. Our results demonstrate MP may be more efficiently and accurately isolated from whole blood in a time-and cost-effective manner. It shall prove an ideal tool in characterizing MP in multiple center clinical studies.

Influence of Sample Collection and Storage on the Detection of Platelet Factor 4–Heparin Antibodies

Heparin-induced thrombocytopenia is a life threatening thrombotic disorder caused by antibodies to platelet factor 4 (PF4) and heparin. Commercial immunoassays are frequently used for the detection of PF4-heparin antibodies, and several studies have reported that higher antibody titers are more frequently associated with adverse events. It is not known if conditions involving sample preparation and/or storage affect the operational characteristics of PF4-heparin immunoassays. We compared the detection of PF4-heparin antibodies from 48 patient samples collected concordantly in serum separator tubes or tubes containing EDTA or sodium citrate. We also examined the effects of extended sample storage on whole blood collected in serum separator, EDTA, or citrate tubes at 4 degrees C for up to 96 hours on antibody detection. We noted that serum or plasma anticoagulated with sodium citrate or EDTA yielded comparable results. In addition, we could not demonstrate any significant sample deterioration after storage at 4 degrees C in any medium for up to 4 days. These findings suggest that PF4-heparin antibodies are largely insensitive to the effects of sample preparation and storage.

Functional Linkage between NOXA and Bim in Mitochondrial Apoptotic Events

NOXA is a BH3-only protein whose expression is induced by certain p53-depenent or independent apoptotic stimuli. Both NOXA and Bim are avid binders of Mcl-1, but a functional linkage between these BH3-only proteins has not yet been reported. In this study, we demonstrate that Mcl-1 binding of endogenously induced NOXA interferes with the ability of Mcl-1 to efficiently sequester endogenous Bim, as Bim is displaced from its complex with Mcl-1. Induced NOXA significantly enhances the UV sensitivity of cells, and the ensuing mitochondrial depolarization is entirely abrogated by Bim knockdown. These results demonstrate a Mcl-1-mediated cross-talk between endogenous NOXA and Bim that occurs upstream of the Bak/Bax-dependent execution of UV-induced mitochondrial depolarization. The current findings demonstrate that the mitochondrial response to an induced expression of NOXA is executed by endogenous Bim and suggest a plausible mechanism for the observed NOXA-Bim linkage.