Phosphate-buffered Saline Albumin Research Articles

The development and application of glyco-probes for bovine serum albumin

Two glyco-probes, GFP and GFN, which are derived from the condensation of 3-acetyl-5-C-(2,3-dihydroxy-1,4-anhydro-β-D-erythro-tetrofuranosyl)-2-methylfuran with benzaldehyde and 1-naphthaldehyde, respectively, are designed and synthesized. Of these two probes, GFP displays a selective and sensitive switch-on fluorescence response towards bovine serum albumin in phosphate-buffered saline over a wide pH range. Moreover, the fluorescence of this probe displays a good linear relationship with the bovine serum albumin concentration ranging from 0 to 0.8 mg/mL, and has a limit of detection as low as 3.7 nM. The stoichiometric ratio of bovine serum albumin /GFP is 1:1. This research paves the way for the development of glyco-probes for bovine serum albumin sensing.

Effect of Saturation Pulse Duration and Power on pH-weighted Amide Proton Transfer Imaging: A Phantom Study.

Amide proton transfer (APT) imaging may detect changes in tissues' pH based on the chemical exchange saturation transfer (CEST) phenomenon, and thus it may be useful for identifying the penumbra in ischemic stroke patients. We investigated the effect of saturation pulse duration and power on the APT effect in phantoms with different pH values. Five samples were prepared from a 1:10 solution of egg-white albumin in phosphate-buffered saline at pH 6.53-7.65. The APT signal intensity (SI) was defined as asymmetry of the magnetization transfer ratio at 3.5 ppm. We measured the APT SIs in the egg-white albumin samples of different pH values with saturation pulse durations of 0.5, 1.0, 2.0, and 3.0 sec and saturation pulse powers of 0.5, 1.5, and 2.5 μT. The relative change in the APT SI in relation to the saturation duration and power at different pH values was defined as follows: (APT SI each saturation pulse - APT SI shortest or weakest pulse)/APT SIshortest or weakest pulse. The dependence of the APT SI on pH and the relative change in the APT SI were calculated as the slope of the linear regression. The lower the pH, the larger the relative change in the APT SI, due to the change in saturation pulse duration and power. The APT SI was highly correlated with the pH at all saturation pulse durations and powers. The influence of saturation duration and power on the APT effect was greater at lower pH than higher pH. The combination of saturation pulse ≥ 1.0 s and power ≥ 1.5 μT was useful for the sensitive detection of changes in APT effects in the egg-white albumin samples with different pH values.

Container Closure Integrity Test Using Frequency Modulation Spectroscopy Headspace Analysis with Carbon Dioxide as a Tracer Gas.

As described in USP <1207>, the container closure integrity (CCI) of a pharmaceutical package must be maintained throughout the product lifecycle to ensure sterility and stability. Current CCI test methods can be time-consuming, destructive, and lack the required sensitivity. This study presents a novel, fast, and nondestructive method for CCI testing that uses carbon dioxide as a tracer gas under effusive pressure conditions. Two types of defects were tested: laser-drilled defects located in the glass body (2, 5, and 10 μm nominal diameter) and tungsten wires inserted between the stopper and the landing seal of the vial (41, 64, and 80 μm outer diameter). During each test session, vials were placed in a pressure vessel, isolated from ambient conditions, and pressure-cycled by first pulling a vacuum and then applying an overpressure of pure carbon dioxide gas. After being exposed to 20 psig (34.7 psia) of carbon dioxide for 30 min, the overpressure was released and the vials were measured on an FMS-Carbon Dioxide Headspace Analyzer. This headspace gas analyzer utilizes a tunable diode laser absorption spectroscopy technique that employs frequency modulation to enhance measurement sensitivity. An increase of ≥1 torr in the headspace carbon dioxide content after completion of the pressure cycling procedure was intended to serve as confirmation of leak detection. All empty vials with either a 2 µm laser-drilled defect or 41 µm wire (effective defect size ∼2 µm), or greater, at the stopper-seal interface were detected by this method. Furthermore, vials filled with 1 mg/mL bovine serum albumin in phosphate-buffered saline containing a 5 μm laser-drilled defect below the liquid level or a 64 µm wire (effective defect size ∼6.1 µm), or greater, at the stopper-seal interface (defect above the liquid level) were detected. This test can be used for a wide variety of vial types and headspace compositions.

Experimental determination of second virial coefficients by small-angle X-ray scattering: a problem revisited

This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions-the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20-100g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein-cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory [Formula: see text] still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source-protein self-interactions and/or protein-cosolute contributions.

Comparison of Peroxidase‐Anti‐Peroxidase and Avidin‐Biotin‐Peroxidase Complex (ABC) Immunohistochemical Staining Procedures for Tryptophan Hydroxylase in Rat Brain

The neurotransmitter serotonin (5‐hydroxytryptamine; 5‐HT) plays a key role in sleep, feeding behavior, motor activity and, importantly, mood and the mechanism of action of antidepressant drugs. Immunohistochemistry for tryptophan hydroxylase (TpOH), the synthetic enzyme for 5‐HT, can be used to identify 5‐HT neurons in the central nervous system. As part of a project examining 5‐HT in brain function in the rat, we compared 2 indirect methods of visualizing primary antibodies against TpOH, a peroxidase‐anti‐peroxidase (PAP) method and the more sensitive avidin‐biotin‐peroxidase (ABC) procedure.A cryostat microtome was used to cut 40 micron sections from brains from rats transcardially perfused with 0.9% saline and 4% p‐formaldehyde in 0.1 M phosphate buffer (PB). Brains were post‐fixed 24 h in p‐formaldehyde and cryoprotected in 20% sucrose in 0.1 M PB. Sections were washed in phosphate‐buffered saline (PBS) before and between all steps. Tissues were treated with 0.4% H2O2 in methanol for 20 min to quench endogenous peroxidase activity and 1% sodium borohydride in 0.1 M PB for 30 min to enhance permeability of the tissue and improve staining quality. Sections were then treated with 1% normal donkey (PAP method) or horse (ABC method) serum in PBS with 0.2% Triton X‐100 detergent for 1 h and then 0.8% bovine serum albumin in PBS for 40 min. Tissues were incubated for 48 h in mouse monoclonal anti‐TpOH antibody (Sigma, 1:1000) in PBS with 0.3% Triton X‐100 and serum. Some sections were incubated without the primary antibody (omit negative controls). For the PAP protocol, tissues were treated with donkey anti‐mouse antibody (Jackson; 1:100) and then mouse PAP (Jackson; 1:400), both in PBS with 0.3% Triton X‐100 for 1 h. For the ABC protocol, tissues were treated with biotinylated horse anti‐mouse antibody and then avidin‐biotin‐peroxidase complex (Vector Elite kit), both in PBS with 0.3% Triton X‐100 and serum for 1 h. All sections were reacted with 0.0005% diaminobenzidine with 0.0001% H2O2 for 45 – 120 sec and mounted on gelatin‐coated slides that were coverslipped with DPX. Stained and negative control sections from both methods were examined using standard light microscopy.Staining using the PAP method was faint, homogenous and only slightly darker than negative controls. Individual neurons could not be identified. Increasing concentrations (1:500, 1:250, 1:100) of anti‐TpOH antibody had no effect on specific labeling but slightly increased background staining. This was surprising because the exact same reagents and PAP procedure has produced outstanding staining of tyrosine hydroxylase in brainstem catecholamine neurons (e.g., Muma et al., Exp. Neurology, 168:135–143, 2001). In contrast, labeling of TpOH using the ABC method was excellent. TpOH immunoreactivity was highly visible in the somata and primary dendrites of neurons in the dorsal and median raphe nuclei. These results add to and amplify the enhanced staining quality of the ABC procedure over the PAP method in formaldehyde‐fixed rat brain sections.Support or Funding InformationSupported by the School of Biological Sciences and the Department of Psychology, Illinois State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of the viability of cultured human cells in suspensions

The success of cell therapy is directly related to the viability of cells used for transplantation. The cells used for transplantation are in some cases injected in suspension. However, the optimal conditions for the preservation of cell viability upon the preparation and storage of cell suspensions for transplantation have not been defined yet. The aim of the present work consisted in the selection of optimal conditions for the storage of suspensions of human submandibular salivary gland cells, differentiated cells of the submandibular salivary gland, and dermal fibroblasts in biocompatible solutions. Standard procedures of cell isolation and cultivation were used in the study. An automatic cell counter from BioRad was used to count the cells, and viability of the cells was assessed using staining with 4% Trypan Blue. The biocompatible solutions tested included phosphate-buffered saline, physiological saline for injections, and a 2% solution of human albumin in phosphate-buffered saline. The study showed that the human cells under investigation remained viable in suspension at both +4°С and +25°С for at least 24 hours, regardless of the carrier solution used. The highest content of viable cells of the salivary gland (more than 50%) at both temperatures examined was observed when cells were suspended in phosphate-buffered saline. However, the adhesive and proliferative properties of the salivary gland cells were better preserved at +4°С in case of 24 hours of incubation under the conditions described above. Fibroblasts maintained in physiological saline formed a homogeneous single-cell suspension that remained stable for 30 hours at +4°С; virtually no loss of cell viability was observed. The addition of 2% albumin resulted in a decrease of the viability of fibroblasts. Thus, storage and transportation in phosphate- buffered saline at +4°С can be recommended for suspensions of cells of the human submandibular salivary gland, whereas human fibroblast suspensions should be maintained at +4°С in physiological saline.

A Highly Sensitive Liquid Chromatography–Electrospray Ionization–Time of Flight/Mass Spectrometric Assay for the Quantitation of 4-Beta-Hydroxycholesterol and Its Application to in vivo Cytochrome P450 3a Induction by AGM-130

A liquid chromatography–electrospray ionization-time-of-flight/mass spectrometry (LC–ESI-TOF/MS) method was developed for the determination of 4-beta-hydroxycholesterol (4β-HC) in rat plasma, to evaluate cytochrome P450 3A (CYP3A) induction liability of a novel cyclin dependent kinases (CDKs) inhibitor, AGM-130. The method consisted of sample preparation without derivatization reagents for high sensitivity such as picolinyl ester, which could be labor-intensive and time consuming. The 4% bovine serum albumin in phosphate-buffered saline as a surrogate matrix was used in the calibration curve and quality control samples for the determination of 4β-HC. Stable isotope-labeled (SIL) 4β-HC (d7-4β-HC) was used as an internal standard. A quadratic regression (weighted 1/concentration), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 3.01–2220 ng/mL for 4β-HC. For quality control samples at 15.0, 165, and 1820 ng/mL from the qualification experiment, the within-run accuracy ranged from 95% to 105% with precision values ≤10% for 4β-HC. This novel LC–ESI-TOF/MS method was successfully applied to evaluate the potential of CYP3A induction by 5′-hydroxy-5-nitro-indirubin oxime (AGM-130) in rat.

Surface modification of YAG:Ce3+ nanoparticles by poly(acrylic acid) and their biological application

Yttrium aluminum garnet Y3Al5O12 (YAG):Ce3+ nanoparticles prepared by glycothermal method are modified with poly(acrylic acid) (PAA). This surface modification enables YAG:Ce3+ nanoparticles to be well dispersed in phosphate-buffered saline (PBS) without any serious aggregation. YAG:Ce3+ nanoparticles emit yellowish green light under the excitation of blue light. This fluorescent property does not change after the modification of PAA. Streptavidin (SA) is immobilized on the surface of PAA-modified YAG:Ce3+ nanoparticles by using carboxyl groups of PAA as cross-linking sites. We demonstrate the quantitative analysis of bovine serum albumin in PBS by SA-immobilized YAG:Ce3+ nanoparticles.

LC–ESI-MS/MS quantification of 4β-hydroxycholesterol and cholesterol in plasma samples of limited volume

A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500ng/ml and 50 to 2000μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor–product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis.The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC–ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.

ATP secretion in the male reproductive tract: essential role of CFTR

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume expanded mice

Pivotal Role for α1-Antichymotrypsin in Skin Repair

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.

Inositol Hexakisphosphate Kinases Induce Cell Death in Huntington Disease

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy.

Stability of Recombinant Human Thyrotropin Potency Based on Bioassay in FRTL-5 Cells

Recombinant human thyrotropin (rhTSH; Thyrogen®) is approved for use in a 0.9 mg dose/day for 2 consecutive days for diagnosis and treatment of differentiated thyroid cancer. It is recommended that it be injected immediately after reconstitution in the distilled water diluent supplied by the manufacturer. However, Thyrogen has been used off-label in doses less than the standard 0.9 mg dose for stimulation of radioiodine uptake in the treatment of multinodular goiter. To determine whether the biologic activity of Thyrogen can be preserved after dilution, we designed experiments to assess the biologic stability of Thyrogen under different durations and temperatures of storage. rhTSH was diluted in 1% bovine serum albumin in phosphate-buffered saline to a concentration of 0.9 mg /mL and further diluted to 0.1 mg/mL. Aliquots of 0.5 mL were stored at room temperature, 4°C, -11°C, and -60°C for various lengths of time. In addition, rhTSH aliquots were also subjected to incubation for 1 hour at 50°C and to 10 cycles of freezing in dry ice alternating with thawing at 37°C. Bioassays were performed in FRTL-5 cells. rhTSH was added to the media at a final concentration of either 5 ng/mL or 20 ng/mL, and the cells were then incubated for 48 hours. Potency was assessed by measurement of ¹²⁵I-iodide uptake in comparison to cells treated with perchlorate to block iodide uptake. Samples were immunoassayed at day 185 of storage. Samples stored at 4°C, -11°C, -60°C, and room temperature retained activity after storage periods of up to 204 days. Samples subjected to 10 freeze-thaw cycles or heated to 50°C for 1 hour retained full biologic activity. Immunoassay at day 185 showed no difference in immunoactivity in relation to the storage condition. rhTSH kept at 4°C, -11°C, -60°C, and room temperature maintained good biologic potency for more than 6 months of storage when tested in vitro, indicating that the biologic activity is very stable. However, altered sialylation occurring during storage could have altered the half-life of rhTSH. Nevertheless, the data provide reassurance that storage in the cold for a few months does not result in significant loss of biologic activity.

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Selenium Compounds Activate Early Barriers of Tumorigenesis

Selenium chemoprevention by apoptosis has been well studied, but it is not clear whether selenium can activate early barriers of tumorigenesis, namely senescence and DNA damage response. To test this hypothesis, we treated normal and cancerous cells with a gradient concentration of sodium selenite, methylseleninic acid and methylselenocysteine for 48 h, followed by a recovery of 1-7 days. Here we show that selenium compounds at doses of </=LD(50) can induce cellular senescence, as evidenced by the expression of senescence-associated beta-galactosidase and 5-bromo-2-deoxyuridine incorporation, in normal but not cancerous cells. In response to clastogens, the ataxia telangiectasia mutated (ATM) protein is rapidly activated, which in turn initiates a cascade of DNA damage response. We found that the ATM pathway is activated by the selenium compounds, and the kinase activity is required for the selenium-induced senescence response. Pretreatment of the MRC-5 non-cancerous cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl suppresses the selenium-induced ATM activation and senescence. Taken together, the results suggest a novel role of selenium in the activation of early tumorigenesis barriers specific in non-cancerous cells, whereby selenium induces an ATM-dependent senescence response that depends on reactive oxygen species.

Src Induces Podoplanin Expression to Promote Cell Migration

Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration.

Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest

Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G(1) to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage.

Variability in the measurement of lipoprotein(a) in the British Isles

Elevated Lipoprotein(a) concentrations are a risk factor for coronary heart disease; however, methodological problems have prevented its introduction to routine clinical practice. Thirty-six laboratories each assayed 20 samples (the same 20 in each laboratory) using two different Lp(a) kits per laboratory, randomly assigned from the total of 12 used in the study. The duplicate error, i.e. the error between-duplicate analyses for each sample, for all kits was small, indicating all kits had a good precision for all the assays. However, there was a very large variation between the kits in the Lp(a) concentration assigned to a sample that could be over 100%. All methods showed a negative or positive bias as the concentration of Lp(a) increased. Most worryingly, as used in this study, several Lp(a) kits detected Lp(a) in a solution of 5% bovine serum albumin in phosphate-buffered saline. The between-laboratory variation in Lp(a) concentration measured using the same kit was very large, e.g. for a sample with a mean concentration of 78.8 mg/dL Lp(a) the between-laboratory variation was 29.7 mg/dL (37.7%). Even with samples with a relatively low Lp(a) concentration of 16.0 mg/dL had a between-laboratory variation of 12.3 mg/dL (76.8%). There is wide variability in reported Lp(a) concentrations, assayed in the same sample, using different Lp(a) assays. At the present time these differences prevent the use of Lp(a) as a routine diagnostic tool.