Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

A novel and accurate microfluidic assay of CD62L in bladder cancer serum samples.

We report a low-cost, sensitive, bead-based electrochemical immunoarray for soluble L-selectin (or CD62L protein), a potential biomarker for staging bladder cancer. We used a semi-automated modular microfluidic array with online antigen capture on superparamagnetic beads, which were subsequently delivered to a detection chamber housing multiple sensors. The assay was designed to accurately detect CD62L in diluted serum with a limit of detection (LOD) of 0.25 ng mL-1 and a dynamic range of 0.25-100 ng mL-1. The microfluidic array gave significantly better accuracy and higher sensitivity than a standard ELISA kit, which was shown to be subject to significant systematic error at high and low concentration ranges. 31 serum samples from patients with varying grades of bladder cancer and cancer-free controls were analyzed by the immunoarray and ELISA, and the CD62L levels correlated. This work establishes a new accurate assay for determining CD62L levels and highlights the potential of this protein as a biomarker for detecting locoregional progression of bladder cancer.

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

Oxidation of DNA by reactive oxygen species (ROS) yields 8-oxo-7,8-dihydroguanosine (8-oxodG) as primary oxidation product, which can lead to downstream G to T transversion mutations. DNA mutations are nonrandom, and mutations at specific codons are associated with specific cancers, as widely documented for the p53 tumor suppressor gene. Here, we present the first direct LC-MS/MS study (without isotopic labeling or hydrolysis) of primary oxidation sites of p53 exon 7. We oxidized a 32 base pair (bp) double-stranded (ds) oligonucleotide representing exon 7 of the p53 gene. Oxidized oligonucleotides were cut by a restriction endonuclease to provide small strands and enable positions and amounts of 8-oxodG to be determined directly by LC-MS/MS. Oxidation sites on the oligonucleotide generated by two oxidants, catechol/Cu2+/NADPH and Fenton's reagent, were located and compared. Guanines in codons 243, 244, 245, and 248 were most frequently oxidized by catechol/Cu2+/NADPH with relative oxidation of 5.6, 7.2, 2.6, and 10.7%, respectively. Fenton's reagent oxidations were more specific for guanines in codons 243 (20.3%) and 248 (10.4%). Modeling of docking of oxidizing species on the ds-oligonucleotide were consistent with the experimental codon oxidation sites. Significantly, codons 244 and 248 are mutational "hotspots" in nonsmall cell and small cell lung cancers, supporting a possible role of oxidation in p53 mutations leading to lung cancer.

Microfluidic array for simultaneous detection of DNA oxidation and DNA-adduct damage.

Exposure to chemical pollutants and pharmaceuticals may cause health issues caused by metabolite-related toxicity. This paper reports a new microfluidic electrochemical sensor array with the ability to simultaneously detect common types of DNA damage including oxidation and nucleobase adduct formation. Sensors in the 8-electrode screen-printed carbon array were coated with thin films of metallopolymers osmium or ruthenium bipyridyl-poly(vinylpyridine) chloride (OsPVP, RuPVP) along with DNA and metabolic enzymes by layer-by-layer electrostatic assembly. After a reaction step in which test chemicals and other necessary reagents flow over the array, OsPVP selectively detects oxidized guanines on the DNA strands, and RuPVP detects DNA adduction by metabolites on nucleobases. We demonstrate array performance for test chemicals including 17ß-estradiol (E2), its metabolites 4-hydroxyestradiol (4-OHE2), 2-hydroxyestradiol (2-OHE2), catechol, 2-nitrosotoluene (2-NO-T), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 2-acetylaminofluorene (2-AAF). Results revealed DNA-adduct and oxidation damage in a single run to provide a metabolic-genotoxic chemistry screen. The array measures damage directly in unhydrolyzed DNA, and is less expensive, faster, and simpler than conventional methods to detect DNA damage. The detection limit for oxidation is 672 8-oxodG per 106 bases. Each sensor requires only 22 ng of DNA, so the mass detection limit is 15 pg (∼10 pmol) 8-oxodG.

Altered leukotriene B4 metabolism in CYP4F18-deficient mice does not impact inflammation following renal ischemia.

Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B4, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B4 in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B4, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B4 to 19-hydroxy leukotriene B4, and to a lesser extent 18-hydroxy leukotriene B4, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia-reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B4 in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia-reperfusion in mice.

Macrophage migratory inhibitory factor promotes bladder cancer progression via increasing proliferation and angiogenesis.

Macrophage migratory inhibitory factor (MIF) is a proinflammatory cytokine shown to promote tumorigenesis. Using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model of bladder cancer, we previously showed that MIF knockout mice display decreased angiogenesis and invasion compared with wild-type. This study examines the role of MIF in bladder cancer via use of oral inhibitors of MIF. In vitro, high-grade bladder cancer cells were treated with recombinant human MIF +/- (rhMIF+/-) inhibitor. Measurements included cell counts, proliferation by (3)H-thymidine incorporation (TdR), extracellular signal-regulated kinase (ERK) phosphorylation by western blot analysis, messenger RNA (mRNA) expression by quantitative PCR and protein secretion by enzyme-linked immunosorbent assay. Treatment with rhMIF increased ERK phosphorylation, cell counts, TdR and mRNA expression and protein secretion of vascular endothelial growth factor, which were blocked by specific inhibitors of ERK and MIF. In vivo, 3-month-old male C57Bl/6 mice were given BBN for 22 and 16 weeks in study 1 and study 2, respectively. Mice (n = 8-10 per group) were gavaged with vehicle or doses of MIF inhibitors daily from weeks 16-22 in both studies. Average bladder weights, reflecting tumor mass, tumor stage/burden, mitotic rate and proliferation indices, and microvessel densities were reduced in inhibitor groups versus controls. In summary, MIF promotes bladder cancer via increasing cell proliferation and angiogenesis and oral inhibitors of MIF may prove useful in treatment of this disease.

Evaluation of a Required Vertical Point-of-Care Ultrasound Curriculum for Undergraduate Medical Students.

Introduction Point-of-care ultrasound training beginning in undergraduate medical education reinforces anatomy and physical examination skills and enhances clinical care. Implementation in an overcrowded curriculum requires strategic planning to overcome barriers including lack of faculty and equipment. Using Kern's six-step model as a framework, our study question was whether a longitudinal point-of-care ultrasound curriculum threaded through four years of medical school and using a novel combination of evidence-based strategies was feasible, acceptable, and resulted in students achieving ultrasound competencies by graduation. Materials and methods From 2016 to 2020, a required, vertical point-of-care ultrasound curriculum was created across all four undergraduate medical education class years, spearheaded by a single ultrasound fellowship-trained emergency physician with support from two basic anatomy faculty. We utilized strategies including handheld ultrasound devices, near-peer teaching, flipped classroom with virtual learning modules, staggered station rotations, and gamification to optimize student-instructor ratios and faculty time. Surveys and timed objective structured clinical assessments evaluated the curriculum. Results Students from the class of 2022 (n=99, 100% of class) participated in all curricular elements. Senior students answered more survey knowledge questions correctly when compared to pretest questions answered by first- and second-year students. Among 84 students who completed the survey, 75 (89%) rated their ultrasound curriculum as superior or above average. Objective structured clinical examination scores recorded for 53 students (54% of the class) demonstrated students correctly identified a median of 11-18 structures (interquartile range: 9.5-13) using point-of-care ultrasound. Conclusion Evidence-based strategies allowed faculty to develop a four-year required ultrasound curriculum that was highly acceptable by students and improved their knowledge and skills at graduation. At low cost and with few faculty, this program has been sustained for over six years.

Detecting cancer metastasis and accompanying protein biomarkers at single cell levels using a 3D-printed microfluidic immunoarray.

A low-cost microfluidic microarray capable of lysing cells and quantifying proteins released after lysis was designed and 3D-printed. The array lyses cells on-chip in lysis buffer augmented with a 2s pulse of a sonic cell disruptor. Detection of desmoglein 3 (DSG3), a metastatic biomarker for head and neck squamous cell carcinoma (HNSCC), along with two accompanying HNSCC biomarkers from a single cell lysate of oral cancer cell cultures was demonstrated. A lysis chamber and reagent compartments deliver sample and reagents into detection chambers decorated with capture antibodies immobilized onto inner walls coated with a highly swollen 3D chitosan hydrogel film. Sandwich immunoassays are achieved when captured analytes labeled with biotinylated secondary antibodies, which then capture streptavidin-poly [horse radish peroxidase] (Poly-HRP). Subsequent delivery of super-bright femto-luminol with H2O2 generates chemiluminescence captured with a CCD camera. DSG3 is membrane-bound protein in HNSCC cells of invaded lymph nodes, vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-C (VEGF-C) were positive controls overexpressed into the HNSCC culture medium. Beta-tubulin (ß-Tub) was used as a loading control to estimate the number of cells in analyzed samples. Limits of detection (LOD) were 0.10 fg/mL for DSG3, and 0.20 fg/mL for VEGF-A, VEGF-C and ß-Tub. Three orders of magnitude semilogarithmic dynamic ranges were achieved. VEGF-A showed high in-cell expression, but VEGF-C had low levels inside cells. The very low LODs enabled quantifying these proteins released from single cells. Strong correlation between results from on-chip cell lysis, conventional off-line lysis and ELISA confirmed accuracy.

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors.

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.

SATB1 and bladder cancer: Is there a functional link?

PURPOSE: SATB1, a global genome organizer, has been shown to play a role in the development and progression of some solid tumors, but its role in bladder cancer is undetermined. Moreover, there is conflicting data about the role of SATB1 in other tumors. This study was initiated to assess a potential role for SATB1 with the hypothesis that SATB1 acts as a tumor promoter in bladder cancer. MATERIALS AND METHODS: We evaluated SATB1 expression in bladder cancer cell lines (HTB-5, HTB-9) and compared them to a benign urothelial cell line (UROtsa). Short-hairpin RNA was used to silence SATB1 in multiple cell lines, and cell death and cell proliferation were assessed using multiple assays. RESULTS: SATB1 expression was increased significantly in all cancer cell lines compared to benign urothelial cells. SATB1 expression was knocked down by short-hairpin RNA and functional outcomes, including cell number, cell-cycle arrest, cell viability, and apoptosis after cisplatin treatment, were measured. Surprisingly, knockdown of SATB1 in 2 high-grade cancer cell lines showed opposing functional roles. Compared to the non-silencing control, HTB-5 cells, showed decreased cellular proliferation and increased sensitivity to cisplatin, whereas HTB-9 cells, showed increased cell numbers and increased resistance to cisplatin. CONCLUSION: We conclude that our results in bladder cancer are consistent with the conflicting data reported in other cancers, and that SATB1 might have different roles in cancer dependent on genetic background and stage of the cancer.

The Role of Pyruvate Dehydrogenase Kinase-4 (PDK4) in Bladder Cancer and Chemoresistance.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1-PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0-G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004-12. ©2018 AACR.

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32 bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent SN2 coupling.

Biodistribution of gold nanoparticles in BBN-induced muscle-invasive bladder cancer in mice.

Bladder-sparing options are being developed for muscle-invasive bladder cancer in place of radical cystectomy, including the combination of chemotherapy and radiation therapy. We reasoned that improving the radiotherapy component of chemoradiation could improve the control of locally advanced disease. Previously, we showed that gold nanoparticles (AuNPs) are potent enhancers of radiation therapy. We hypothesized that if AuNPs were to preferentially localize to bladder tumors, they may be used to enhance the radiation component of muscle-invasive bladder tumor therapy. Mice were treated with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 17, 20, and 22 weeks - long enough to induce muscle-invasive tumors. Mice were then anesthetized and injected intravenously with 1.9 nm AuNPs of which most were rapidly cleared from the blood and excreted after a 30-50 minute residence time in the bladder. We found AuNPs distributed throughout the bladder wall, but most of the AuNPs were associated with the stroma surrounding the tumor cells or extracellular keratin produced by the tumor cells. There were relatively few AuNPs in the tumor cells themselves. The AuNPs therefore localized to tumor-associated stroma and this tumor specificity might be useful for specific X-ray dose enhancement therapy of muscle-invasive bladder carcinomas.

CHEMICAL SELECTIVITY OF NUCLEOBASE ADDUCTION RELATIVE TO IN VIVO MUTATION SITES ON EXON 7 FRAGMENT OF P53 TUMOR SUPPRESSOR GENE.

Damage to p53 tumor suppressor gene is found in half of all human cancers. Databases integrating studies of large numbers of tumors and cancer cell cultures show that mutation sites of specific p53 codons are correlated with specific types of cancers. If the most frequently damaged p53 codons in vivo correlate with the most frequent chemical damage sites in vitro, predictions of organ-specific cancer risks might result. Herein, we describe LC-MS/MS methodology to reveal codons with metabolite-adducted nucleobases by LC-MS/MS for oligonucleotides longer than 20 base pairs. Specifically, we used a known carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) to determine the most frequently adducted nucleobases within codons. We used a known sequence of 32 base pairs (bp) representing part of p53 exon 7 with 5 possible reactive hot spots. This is the first nucleobase reactivity study of a double stranded DNA p53 fragment featuring more than 20 base pairs with multiple reactive sites. We reacted the 32 bp fragment with benzo[a]pyrene metabolite BPDE that undergoes nucleophilic substitution by DNA bases. Liquid chromatography-mass spectrometry (LC-MS/MS) was used for sequencing of oligonucleotide products from the reacted 32 bp fragment after fragmentation by a restriction endonuclease. Analysis of the adducted p53 fragment compared with unreacted fragment revealed guanines of codons 248 and 244 as most frequently targeted, which are also mutated with high frequency in human tumors. Codon 248 is mutated in non-small cell and small cell lung, head and neck, colorectal and skin cancer, while codon 244 is mutated in small cell lung cancer, all of which involve possible BDPE exposure. Results suggest the utility of this approach for screening of adducted p53 gene by drugs and environmental chemicals to predict risks for organ specific cancers.

Elucidating Organ-Specific Metabolic Toxicity Chemistry from Electrochemiluminescent Enzyme/DNA Arrays and Bioreactor Bead-LC-MS/MS.

Human toxic responses are very often related to metabolism. Liver metabolism is traditionally studied, but other organs also convert chemicals and drugs to reactive metabolites leading to toxicity. When DNA damage is found, the effects are termed genotoxic. Here we describe a comprehensive new approach to evaluate chemical genotoxicity pathways from metabolites formed in-situ by a broad spectrum of liver, lung, kidney and intestinal enzymes. DNA damage rates are measured with a microfluidic array featuring a 64-nanowell chip to facilitate fabrication of films of DNA, electrochemiluminescent (ECL) detection polymer [Ru(bpy)2(PVP)10]2+ {(PVP = poly(4-vinylpyridine)} and metabolic enzymes. First, multiple enzyme reactions are run on test compounds using the array, then ECL light related to the resulting DNA damage is measured. A companion method next facilitates reaction of target compounds with DNA/enzyme-coated magnetic beads in 96 well plates, after which DNA is hydrolyzed and nucleobase-metabolite adducts are detected by LC-MS/MS. The same organ enzymes are used as in the arrays. Outcomes revealed nucleobase adducts from DNA damage, enzymes responsible for reactive metabolites (e.g. cyt P450s), influence of bioconjugation, relative dynamics of enzymes suites from different organs, and pathways of possible genotoxic chemistry. Correlations between DNA damage rates from the cell-free array and organ-specific cell-based DNA damage were found. Results illustrate the power of the combined DNA/enzyme microarray/LC-MS/MS approach to efficiently explore a broad spectrum of organ-specific metabolic genotoxic pathways for drugs and environmental chemicals.

Increased expression of L-selectin (CD62L) in high-grade urothelial carcinoma: A potential marker for metastatic disease.

INTRODUCTION: L-Selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leukocytes with a primary function of directing leukocyte migration and homing of lymphocytes to lymph nodes. In a gene expression microarray study comparing laser-captured microdissected high-grade muscle-invasive bladder cancer (MIBC) without prior treatment and low-grade bladder cancer (LGBC) human samples, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. METHODS: Unfixed fresh and formalin-fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the University of Connecticut Health Center tumor bank. Tumor cells were isolated from frozen tumor tissue sections by laser-captured microdissected followed by RNA isolation. Quantitative polymerase chain reaction was used to validate the level of CD62L transcripts. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine database. RESULTS: Immunostaining showed a uniformly higher expression of CD62L in MIBC specimens vs. LGBCs specimens. Further, CD62L localization was seen in foci of metastatic tumor cells in lymph node specimens from patients with high-grade MIBC and known nodal involvement. Up-regulated expression of CD62L was also observed by flow cytometric analysis of freshly isolated tumor cells from biopsies of high-grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher in serum samples from patients with high-grade metastatic vs. high-grade nonmetastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. CONCLUSION: These data confirm the expression of CD62L on urothelial carcinoma cells and suggest that CD62L may serve as biomarker to predict the presence of or risk for developing metastatic disease in patients with bladder cancer.

Salt- and glyphosate-induced increase in glyoxalase I activity in cell lines of groundnut (Arachis hypogaea).

Glyoxalase I (EC 4.4.1.5) activity has long been associated with rapid cell proliferation, but experimental evidence is forthcoming, linking its role to stress tolerance as well. Proliferative callus cultures of groundnut (Arachis hypogaea L. cv. JL24) showed a 3.3-fold increase in glyoxalase I activity during the logarithmic growth phase, correlating well with the data on FW gain and mitotic index. Inhibition of cell division decreased glyoxalase I activity and vice versa, thus further corroborating its role as a cell division marker enzyme. Cell lines of A. hypogaea selected in the presence of high salt (NaCl) and herbicide (glyphosate) concentrations, yielded 4.2- to 4.5-fold and 3.9- to 4.6-fold elevated glyoxalase I activity, respectively, in a dose dependent manner reflective of the level of stress tolerance. The stress-induced increase in enzyme activity was also accompanied by an increase in the glutathione content. Exogenous supplementation of glutathione could partially alleviate the growth inhibition of callus cultures induced by methylglyoxal and d-isoascorbic acid, but failed to recover the loss in glyoxalase I activity due to d-isoascorbic acid. The adaptive significance of elevated glyoxalase I activity in maintaining glutathione homeostasis has been discussed in view of our understanding on the role of glutathione in the integration of cellular processes with plant growth and development under stress conditions.

Ultrasensitive nanostructured immunosensor for stem and carcinoma cell pluripotency gatekeeper protein NANOG.

AIMS: To develop an immunosensor for ultrasensitive detection of the NANOG protein. NANOG regulates pluripotency in stem cells and some cancer cells. This article reports the first electrochemical immunosensor for ultrasensitive detection and absolute quantification of the NANOG protein. The sensor features dense capture antibody-coated gold nanoparticle layers on a pyrolytic graphite underlayer. MATERIALS & METHODS: Two separate multilabel detection strategies were used to achieve moderate and ultra-high sensitivity. RESULTS: Good sensitivity was achieved for NANOG over the concentration range 0.1-160 pg/ml. The moderate-sensitivity approach gave a detection limit of 25 pg/ml, while the ultrasensitive method achieved a 250-fold lower detection limit of 0.1 pg/ml. Amounts of NANOG detected in human embryonic stem cell lysates correlated well with qualitative western blots and mRNA expression. CONCLUSION: The electrochemical gold nanoparticle immunosensor is suitable for measuring NANOG protein expression in stem and carcinoma cell tissue lysates at very low levels.

Accurate DNA fragment sizing by capillary electrophoresis with laser-induced fluorescence array for detection of sequence specificity of DNA damage.

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [...GGCGCGCAG...] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with (32)P end labeling.

Characterization of the biochemical and structural phenotypes of four CYP1B1 mutations observed in individuals with primary congenital glaucoma.

OBJECTIVE: The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule. METHODS: CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize beta-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure. RESULTS: CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4 degrees C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern. CONCLUSION: Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.