Concomitant sensitization to glutaraldehyde and methacrylic monomers among dentists and their patients.

BACKGROUND: A multitude of methacrylic monomers is used in dentistry. Glutaraldehyde (G) is used in dental practice and consumer products as a broad-spectrum antimicrobial agent. The purpose of our study is to evaluate the frequency and the risk of concomitant sensitization to some methacrylic monomers (methyl methacrylate (MMA), triethyleneglycol dimethacrylate (TEGDMA), ethyleneglycol dimethacrylate (EGDMA), 2,2-bis-[4-(2-hydroxy-3-methacrylo-xypropoxy)phenyl]-propane (Bis-GMA), 2-hydroxy-ethyl methacrylate (2-HEMA) and tetrahydrofurfuryl methacrylate (THFMA)) and glutaraldehyde in students of dentistry, students from the dental technician school, dental professionals and dental patients. MATERIAL AND METHODS: A total of 262 participants were included in the study: students of dentistry, students from the dental technician school, dental professionals, and dental patients as a control group. All were patch-tested with methacrylic monomers and glutaraldehyde. The results were subject to the statistical analysis (p < 0.05). RESULTS: Among the group of dental students, the highest frequency of concomitant sensitization was to TEGDMA and G (15.5%). In the group of patients the highest frequency of concomitant sensitization was to EGDMA and G (16.4%). The frequency of concomitant sensitization among dental professionals was much lower, with the highest rate to TEGDMA and G (7.7%), too. We consider the students from the dental technician school, where the exposure to glutaraldehyde is less likely, to be the group at a lesser risk of concomitant sensitization. CONCLUSIONS: Dental students and dental patients could be outlined as groups at the risk of concomitant sensitization to glutaraldehyde and methacrylic monomers. For dental professionals, we assumed an increased risk for concomitant sensitization to TEGDMA and aldehydes that are commonly used in dentistry. We consider the students from the dental technician school to be the group at a lesser risk of concomitant sensitization to glutaraldehyde and methacrylic monomers. Med Pr 2016;67(3):311-320.

Inhibition of circulating dipeptidyl peptidase 4 activity in patients with metastatic prostate cancer.

Cancer is responsible for many deaths and is a major source of healthcare expenditures. The identification of new, non-invasive biomarkers might allow improvement of the direct diagnostic or prognostic ability of already available tools. Here, we took the innovative approach of interrogating the activity of exopeptidases in the serum of cancer patients with the aim of establishing a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease relative to cancer-free littermates. We identified the protease in question as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in the sera of patients with metastatic prostate cancer relative to patients with localized disease or healthy controls. However, no significant differences in DPP4 serum levels were observed, which established the loss of activity as the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity was the result not of post-translational modifications or allosteric changes, but instead of a low-molecular-weight inhibitor. After we adjusted for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity might be a potential blood-based indicator of the presence of metastatic cancer of prostatic origin, either by itself or, more likely, as a means to improve the sensitivity and specificity of existing markers.

Aminopeptidase activities as prospective urinary biomarkers for bladder cancer.

PURPOSE: Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer. EXPERIMENTAL DESIGN: The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential. RESULTS: Nineteen exopeptidases and 21 other proteases were identified in urine and the top-five most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (p ≤ 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898. CONCLUSION AND CLINICAL RELEVANCE: We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intraindividual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.

Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes.

Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI-SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on 'suppressor of tumorigenecity 7' (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me(2))R8, H4(Me(2))R3 and H3(Me(3))K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression.

Isolation and mass spectrometry of specific DNA binding proteins.

A subset of the proteome that binds to specific DNA sequences is at the center of genome function, integrity, and dynamics. We present a detailed protocol that allows the isolation of any specific DNA binding protein and its subsequent identification by mass spectrometry. The procedure involves prefractionation of crude nuclear extract by phosphocellulose (P11) chromatography, followed by a series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format. DNA-affinity capture requires concatamerized and biotinylated ligand, selective salt conditions, and improved competitor DNAs. The amount of protein(s) captured on DNA-magnetic beads is generally sufficient for successful MALDI-TOF and TOF/TOF MS-based protein identification. As an example, we describe the procedures used to isolate and identify four specific transcription factors from 2 x 10(9) promyelocytic NB4 cells.

PU.1 and a TTTAAA element in the myeloid defensin-1 promoter create an operational TATA box that can impose cell specificity onto TFIID function.

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein ("D1BP"), acting through, respectively, proximal (-22/-19) and distal (-62/-59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)alpha/GABPbeta. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking "TTTAAA" element (-32/-27), a "weak" but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a "strong" TATA box ("TATAAA") eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.

Non-homologous end joining, but not homologous recombination, enables survival for cells exposed to a histone deacetylase inhibitor.

on-homologous end joining (NHEJ) and homologous recombination (HR) are pathways that repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, the repair of these breaks is influenced by histone acetylation. Therefore, we tested mammalian cells deleted for NHEJ (Ku80 or DNA Ligase IV) or altered for HR (breast cancer associated gene, Brca2, or Bloom's syndrome, Blm) for sensitivity to trichostatin A (TSA), a histone deacetylase inhibitor that is being investigated as an anti-cancer therapeutic. We show that cells mutated for Ku80 (ku80-/-) or DNA Ligase IV (lig 4-/-), but not cells mutated for Brca2 (brca2lex1/lex2) or Blm (blm(tm3Brd/tm4Brd)), are hypersensitive to TSA in a dose-dependent manner. TSA-induced toxicity stimulates apoptosis and cell cycle checkpoint responses independent of p53, but does not increase phosphorylated histone H2AX (-H2AX) as compared with a clastogenic agent, camptothecin, indicating that the quantity of DSBs is not the primary cause of TSA-induced cell death. In addition, we show that potential anti-cancer drugs (LY-294002 and vanillin) that inhibit the family of phosphatidylinositol 3 kinases that include the NHEJ protein, DNA-PKCS act in synergy with TSA to reduce the viability of HeLa cells in tissue culture presenting the possibility of using the two drugs in combination to treat cancer.

A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation.

Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs.

Delayed-late activation of a myeloid defensin minimal promoter by retinoids and inflammatory mediators.

Alpha-defensin-1 gene expression in promyelocytic HL-60 cells is ('delayed-late' > or =1-2 days) activated by retinoic acid (RA), lipopolysaccharide, tumor necrosis factor-alpha and elevated levels of cAMP. Using stably integrated reporter constructs, we show that this activation is directed through a proximal and distal element within a minimal (-83/+82) def1 promoter, and is mediated by phosphorylation of the associated factors, PU.1 and D1BP, in an inducer-dependent manner. Whereas binding of PU.1 to the proximal element confers cell specificity and relays the effects of most inducers, the selectively enhancing capacity of the distal element for RA- and cAMP-dependent activation is uniquely correlated with D1BP-binding. We propose that D1BP and PU.1 are the end-points of separate pathways.

Affinity capture of specific DNA-binding proteins for mass spectrometric identification.

We describe a general approach for affinity microcapture of site-specific, nucleic acid-binding proteins. The major difficulties to developing this method into a widely applicable protocol derived from the need for a massive enrichment and the inadvertent, extensive binding of nonspecific proteins to the bait. On the basis of a detailed analysis, we propose (i) a one-step fractionation of crude extracts on P11 phosphocellulose, followed by (ii) a discrete series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format, with cobalt magnets, concatamerized and biotinylated ligands, selective salt conditions, and improved competitor DNAs. We also present rules for determining the precise number and order of selections. The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2 x 10(9) cultured leukemia cells. Captured proteins were 10-20,000-fold enriched from the nuclear extract, in a form and amounts that permitted facile MALDI-TOF and TOF/TOF MS-based protein identification. This is 1-2 orders of magnitude better than many previous efforts and in a fraction of the time (approximately 1 factor/week). The method can be applied to any protein that binds DNA, including those with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regulation, and DNA repair with the analytical power of mass spectrometry-based proteomics.

Evaluation of the immunomodulatory activity of Aronia in combination with apple pectin in patients with breast cancer undergoing postoperative radiation therapy.

The aim of the present study was to evaluate the immunomodulatory activity of Aronia in combination with apple pectin in patients with breast cancer in the course of postoperative radiation therapy. Monoclonal antibodies were used to assay specific T cell subsets. Tests were performed prior to and after 26 and 50 Gy of irradiation. The study comprised 42 women (19 to 65 years of age) receiving 15 g of apple pectin in combination with 20 ml of Aronia concentrate (Bioactive Substance Laboratory--Plovdiv) twice daily during postoperative irradiation. Irradiation was performed by a 60Co-Rokus according to individualized treatment schedules. The following T lymphocyte populations were tested--CD3 total T lymphocytes, CD4 helper and inducer T cells, CD8 suppressor and cytotoxic T cells and NK cells. The levels of the polypeptide tissue antigen (TPA), an oncofetal protein, were tested in parallel. The TPA was used to assess treatment outcome in our patients. A group of 25 age-matched women with breast cancer served as controls. Immune status analysis of controls was performed prior to and following postoperative radiation. A total of 880 serum samples were tested. Assays of immunity parameters in the patients receiving Aronia in combination with apple pectin showed that CD4 and CD8 T cell counts increased significantly (P < 0.01 and P < 0.05 respectively). In control patients CD3 T cell levels lowered, the other T cell levels remained unchanged. Initially the number of NK cells was increased in both groups of patients. It remained constant throughout the course of the study. The normal levels of TPA in both groups of patients indicated a good treatment outcome due to the adequacy of surgery and in combination with radiation therapy.