Exosomal survivin facilitates vesicle internalization.

Survivin, a member of the inhibitor of apoptosis (IAP) protein family plays a significant role in cell fate and function. It is significantly overexpressed in tumor cells and has been identified in most cancer cell types. A novel extracellular population has recently been identified and its function is still unknown.

Proteomic Profiling of Serum-Derived Exosomes from Ethnically Diverse Prostate Cancer Patients.

Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved.

Antimetabolite Treatment for Pancreatic Cancer.

Pancreatic cancer is a deadly and aggressive disease. Less than 1% of diagnosed patients survive 5 years with an average survival time of only 4-8 months. The only option for metastatic pancreatic cancer is chemotherapy where only the antimetabolites gemcitabine and 5-fluorouracil are used clinically.

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase.

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment.

Kinetics of 3-nitrotyrosine modification on exposure to hypochlorous acid.

The markers 3-nitrotyrosine and 3-chlorotyrosine are measured as surrogates for reactive nitrogen species and hypochlorous acid respectively, which are both elevated in inflamed human tissues. Previous studies reported a loss of 3-nitrotyrosine when exposed to hypochlorous acid, suggesting that observations of 3-nitrotyrosine underestimate the presence of reactive nitrogen species in diseased tissue (Whiteman and Halliwell, Biochemical and Biophysical Research Communications, 258, 168-172 (1999)). This report evaluates the significance of 3-nitrotyrosine loss by measuring the kinetics of the reaction between 3-nitrotyrosine and hypochlorous acid.

Evidence of yttrium silicate inclusions in YSZ-porcelain veneers.

This report introduces the discovery of crystalline defects that can form in the porcelain veneering layer when in contact with yttria-stabilized zirconia (YSZ). The focus was on dental prostheses and understanding the defects that form in the YSZ/porcelain system; however the data reported herein may have broader implications toward the use and stability of YSZ-based ceramics in general. Specimens were cut from fully sintered YSZ plates and veneering porcelain was applied (<1 mm thick) to one surface and fired under manufacturer's recommended protocol.

Fatigue testing of electron beam-melted Ti-6Al-4V ELI alloy for dental implants.

Customized one-component dental implants have been fabricated using Electron Beam Melting(®) (EBM(®)), which is a rapid prototyping and manufacturing technique. The goal of our study was to determine the effect of electron beam orientation on the fatigue resistance of EBM Ti-6Al-4V ELI alloy. EBM technique was used to fabricate Ti-6Al-4V ELI alloy blocks, which were cut into rectangular beam specimens with dimensions of 25 × 4 × 3 mm, such that electron beam orientation was either parallel (group A) or perpendicular (group B) to the long axis of the specimens.

Kinetics of 3-chlorotyrosine formation and loss due to hypochlorous acid and chloramines.

The persistent activation of innate immune cells in chronic inflammation is gaining recognition as a contributing factor in a number of human diseases. A distinguishing feature of activated leukocytes at sites of inflammation is their production of reactive species such as hypochlorous acid (HOCl). Investigating the role of reactive molecules such as HOCl in inflammation and human disease requires appropriate biomarkers.

Survivin is released from cancer cells via exosomes.

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells.

A modified "cross-talk" between histone H2B Lys-120 ubiquitination and H3 Lys-79 methylation.

Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput.

Impact of sugar pucker on base pair and mispair stability.

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end.

pH-Dependent configurations of a 5-chlorouracil-guanine base pair.

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G).

Base pairing configuration and stability of an oligonucleotide duplex containing a 5-chlorouracil-adenine base pair.

Inflammation-mediated reactive molecules can damage DNA by oxidation and chlorination. The biological consequences of this damage are as yet incompletely understood. In this paper, we have constructed oligonucleotides containing 5-chlorouracil (ClU), one of the known inflammation damage products.

Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry.

The nucleoside analogue 5-aza-2'-deoxycytidine (Decitabine, DAC) is one of several drugs in clinical use that inhibit DNA methyltransferases, leading to a decrease of 5-methylcytosine in newly replicated DNA and subsequent transcriptional activation of genes silenced by cytosine methylation. In addition to methyltransferase inhibition, DAC has demonstrated toxicity and potential mutagenicity, and can induce a DNA-repair response. The mechanisms accounting for these events are not well understood.

Cloning and characterization of Rhodotorula glutinis thymine hydroxylase.

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH.

The interaction between human papillomavirus type 16 and FADD is mediated by a novel E6 binding domain.

High-risk strains of human papillomavirus, such as types 16 and 18, have been etiologically linked to cervical cancer. Most cervical cancer tissues are positive for both the E6 and E7 oncoproteins, since it is their cooperation that results in successful transformation and immortalization of infected cells. We have reported that E6 binds to tumor necrosis factor receptor 1 and to Fas-associated death domain (FADD) and, in doing so, prevents E6-expressing cells from responding to apoptotic stimuli.

Hypochlorous acid damages histone proteins forming 3-chlorotyrosine and 3,5-dichlorotyrosine.

While the last 30 years chronicles an extensive effort to understand the damage to DNA caused by reactive oxygen species (ROS), little research has examined the chemical damage to the histone proteins found in chromatin. Hypochlorous acid (HOCl), the primary product of activated neutrophils, is known to damage both DNA and proteins. This article describes the use of mass spectrometry to quantitate the formation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl, in the core histone proteins.

The Trp-cage: optimizing the stability of a globular miniprotein.

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.

Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2'-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry.

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage.

Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase.

The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors.

Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase.

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens.

Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial.

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects.

Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance.

Overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase) in muscle and adipose tissue of transgenic mice was previously shown to result in insulin resistance and hyperleptinemia. Explanted muscle from transgenic mice was not insulin resistant in vitro, suggesting that muscle insulin resistance could be mediated by soluble factors from fat tissue. To dissect the relative contributions of muscle and fat to hexosamine-induced insulin resistance, we overexpressed glutamine:fructose-6-phosphate amidotransferase 2.

Peptide conformational changes induced by tryptophan-phosphocholine interactions in a micelle.

Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles.

Designing a 20-residue protein.

Truncation and mutation of a poorly folded 39-residue peptide has produced 20-residue constructs that are >95% folded in water at physiological pH. These constructs optimize a novel fold, designated as the 'Trp-cage' motif, and are significantly more stable than any other miniprotein reported to date. Folding is cooperative and hydrophobically driven by the encapsulation of a Trp side chain in a sheath of Pro rings.