Flavonoid inhibitory pharmacodynamics on platelet function in physiological environments

The complex relationship between flavonoid-based nutrition and cardiovascular disease may be dissected by understanding the activities of these compounds in biological systems. The aim of the present study was to explore a hierarchy for the importance of dietary flavonoids on cardiovascular health by examining the structural basis for inhibitory effects of common, dietary flavonoids (quercetin, apigenin, and naringenin) and the plasma metabolite, tamarixetin. Understanding flavonoid effects on platelets in vivo can be informed by investigations of the ability of these compounds to attenuate the function of these cells. Inhibition of platelet function in whole blood and plasma was structure-dependent. The order of potency was apigenin > tamarixetin > quercetin = naringenin indicating that in vivo, important functional groups are potentially a methylated B ring, and a non-hydroxylated, planar C ring. Apigenin and the methylated metabolite of quercetin, tamarixetin significantly reduced thrombus volume at concentrations (5 μM) that suggested their reported physiological levels (0.1-1 μM) may exert low levels of inhibition. Flavonoid interactions with erythrocytes, leukocytes and human serum albumin in whole blood reduce their inhibitory activities against platelet function. The diminished inhibitory activity of flavonoids that we observed in whole blood and plasma indicated that these interactions do not overcome the attenuating effects of these compounds. Furthermore, inhibition of platelet aggregation by flavonoids was enhanced with increases in exposure time, indicating the potential for measurable inhibitory effects during resident plasma times. We conclude that flavonoid structures may be a major influence of their activities in vivo with methylated metabolites and those of flavones being more potent than those of flavonols and flavanones.

The structural effect of Methyl substitution on the binding of Polypyridyl Ru-dppz Complexes to DNA

ABSTRACT: Polypyridyl ruthenium complexes have been intensively studied and possess photophysical properties which are both interesting and useful. They can act as probes for DNA, with a substantial enhancement in emission when bound, and can induce DNA damage upon photoirradiation and therefore, the synthesis and characterization of DNA binding of new complexes is an area of intense research activity. Whilst knowledge of how the binding of derivatives compares to the parent compound is highly desirable, this information can be difficult to obtain. Here we report the synthesis of three new methylated complexes, [Ru(TAP)2(dppz-10-Me).2Cl, [Ru(TAP)2(dppz-10,12-Me2)].2Cl and [Ru(TAP)2(dppz-11-Me)].2Cl, and examine the consequences for DNA binding through the use of atomic resolution X-ray crystallography. We find that the methyl groups are located in discrete positions with a complete directional preference. This may help to explain the quenching behavior which is found in solution for analogous [Ru(phen)2(dppz)]2+ derivatives.

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Investigations into the analytical applications and fundamental chemistry of the chemiluminescent reactions of Tris(2,2'-bipyridyl)ruthenium(III) with certain Papaver Somniferum alkaloids and other related compounds

The reaction of tris(2,2’-bipyridyl)ruthenium(III) (Ru(bipy) ₃³⁺) with various analytes to generate chemiluminescence has been well documented. This investigation sought to undertake a chemiluminometic study of the reactions of Ru(bipy) ₃³⁺ with selected Papaver Somniferum alkaloids and specifically synthesised phenethylamines. The investigation, based on a kinetic study, primarily addressed the effect of varying reaction conditions (pH) on Ru(bipy) ₃³⁺ chemiluminescence production. To monitor these reactions, a batch chemiluminometer was specifically designed, fabricated and automated to conduct an extensive study on the selected compounds of interest. The instrumentation incorporated a custom built reaction cell and comprised an ‘on-line’ sample preparation system with which calibration standards could be automatically prepared. The instrumentation provided both time-independent (peak area) and time-dependent (kinetic profile) information. A novel approach to the stabilisation of Ru(bipy) ₃³⁺ as a chemiluminescencent reagent was also investigated and a recirculating system was employed with the batch chemiluminometer to provide a stable supply of Ru(bipy) ₃³⁺. Codeine, thebaine and 6-methoxy-codeine were the Papaver Somniferum alkaloids selected for this study and several N-methylated and N,N-dimethylated phenethylamines and methoxy-substituted phenetheylamines were also synthesised to investigate the affect of pH on the chemiluminescence emission efficiency. The versatility of the batch chemiluminometer facilitated the kinetic study of numerous analytes over a broad pH range. The exemplary performance of the chemiluminometer as an analytical instrument, was demonstrated by the calibration functions, based on peak area data, which exhibited excellent linearity and sensitivity. The estimated detection limits (3s) for the selected alkaloids were in the range 2 x 10^-9 M to 7 x 10^-9 at pH 5.0 and above, which compared favourably to detection limits for the same compounds determined using FIA. Relative standard deviations (n=5) for peak areas ranged between 1% to 5% with a mean of 3.1% for all calibration standards above 2.5 x 10^-8 M. Correlation between concentration and peak area, irrespective of pH and analyte was excellent, with all but two calibration functions having r-squared values greater than 0.990. The analytical figures of merit exemplified the precision and robustness of the reagent delivery and ‘on-line’ sample preparation, as well as the sensitivity of the system. The employment of the chemiluminometer for the measurement of total chemiluminescence emission (peak area) was in itself a feasible analytical technique, which generated highly reproducible and consistent data. Excellent analytical figures of merit, based on peak area, were similarly achieved for the phenethylamines. The effects of analyte structure on chemiluminescence activity was also investigated for the alkaloids and the phenethylamines. Subtle structural variations between the three alkaloids resulted in either a moderately reduced or enhanced total emission that was two or three fold difference only. A significant difference in reaction kinetics was observed between thebaine and codeine/6-methoxy-codeine, which was dependent upon pH. The time-dependent data, namely the observed rate constants for the initial rise in intensity and for the subsequent decay rate, were obtained by fitting a mathematical function (based on the postulated reaction mechanism) to the raw data. The determination of these rate constants for chemiluminescence reactions highlighted the feasibility for utilising such measurements for quantitative analytical applications. The kinetic data were used to discriminate between analyte responses in order to determine the concentrations of individual analytes in a binary mixture. A preliminary, multi-component investigation performed on a binary mixture of codeine and 6-methoxy-codeine (1:1) successfully determined the concentrations of these individual components using such rate constant measurements. Consequently, variations in kinetics resulted in a significant difference between the relative chemiluminescence response based on peak area measurements and the relative response base on peak height measurements obtained using FIA. With regards to the observed reactivity of secondary amines and tertiary amines, chemiluminescence peak area determinations confirmed the vital role of pH on reaction efficiency, which was governed by structural features and kinetics. The tertiary amines investigated generally produced a greater emission under acidic conditions than the corresponding secondary amines. However, the measured chemiluminescence responses were highly dependent upon pH, with similar peak areas obtained for both amine groups under slightly alkaline conditions.

Controlled modification of the inorganic and organic bricks in an Al-based MOF by direct and post-synthetic synthesis routes

Four new porous CAU-1 derivatives CAU-1–NH2 ([Al4(OH)2(OCH3)4(BDC–NH2)3]·xH2O, BDC–NH22− = aminoterephthalate), CAU-1–NH2(OH) ([Al4(OH)6(BDC–NH2)3]·xH2O), CAU-1–NHCH3 ([Al4(OH)2(OCH3)4(BDC–NHCH3)3]·xH2O) and CAU-1–NHCOCH3 ([Al4(OH)2(OCH3)4(BDC–NHCOCH3)3]·xH2O) all containing an octameric [Al8(OH)4+y(OCH3)8−y]12+ cluster, with y = 0–8, have been obtained by MW-assisted synthesis and post-synthetic modification. The inorganic as well as the organic unit can be modified. Heteronuclear 1H–15N, 1H–13C and homonuclear 1H–1H connectivities determined by solid-state NMR spectroscopy prove the methylation of the NH2 groups when conventional heating is used. Varying reaction times and temperatures allow controlling the degree of methylation of the amino groups. Short reaction times lead to non-methylated CAU-1 (CAU-1–NH2), while longer reaction times result in CAU-1–NHCH3. CAU-1–NH2 can be modified chemically by using acetic anhydride, and the acetamide derivative CAU-1–NHCOCH3 is obtained. Thermal treatment permits us to change the composition of the Al-containing unit. Methoxy groups are gradually exchanged by hydroxy groups at 190 °C in air. Solid-state NMR spectra unequivocally demonstrate the presence of the amino groups, as well as the successful post-synthetic modification. Furthermore 1H–1H correlation spectra using homonuclear decoupling allow the orientation of the NHCOCH3 groups within the pores to be unravelled. The influence of time and temperature on the synthesis of CAU-1 was studied by X-ray powder diffraction, elemental analyses, and 1H liquid-state NMR and IR spectroscopy.

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.

Structure-antimicrobial activity of some natural isocoumarins and their analogues

Three naturally occurring isocoumarins (paepalantine, paepalantine 9-O-beta-D-glucopyranoside and paepalantine 90-beta-D-allopyranosyl(1 -> 6) glucopyranoside) and two semi-synthetic analogues, 9,10-acylated compound and 9-OH-10-methylated compound,. structurally similar to paepalantine, were evaluated for antimicrobial activity using a spectrophotometric microdilution technique. The paepalantine was active against S. aureus, S. epidermidis, and E faecalis. while the other four compounds proved ineffective against all microorganisms tested at concentrations of 500 mu g/ml. Variations in phenolic substitution at OH-9 and/or OH-10 in the paepalantine molecule resulted in compounds without antimicrobial activity. A combination of structural features, two phenolic groups as cathecolic system, forms an oxygenated system arrangement that may reflect the potentially antimicrobial properties of paepalantine. (c) 2004 Elsevier GmbH. All rights reserved.

DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylationsensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.

Fetotoxicity caused by the interaction between zinc and arsenic in mice

Arsenic is an environmental pollutant that induces congenital malformations in experimental models and can contribute to human birth defects. The environmental exposure to arsenic is relatively small when compared with the doses required to cause teratogenicity in mice and other laboratory animals. In order to study the action of zinc in the arsenic-induced teratogenicity, in the present work mice were either pretreated with zinc and later with arsenic or were treated simultaneously with zinc and arsenic in vivo and in vitro. Following administration of arsenate on gestation day 8, pregnant females were killed on the 17th day of gestation; maternal and fetal data were collected by laparotomy and used to calculate reproductive parameters. Fetuses were analyzed for the presence of external malformation and, after the appropriate processing, visceral and skeletal analyses were accomplished. Conceptuses were exposed in whole embryo culture to arsenicals on gestation day 8 (3-6 somite stage). After a 26 h culture period, morphological development was assessed. Neither pretreatment with zinc nor simultaneous administration of zinc prevented arsenic teratogenicity in these experimental models. (C) 2002 Wiley-Liss, Inc.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

LHX6 is a sensitive methylation marker in head and neck carcinomas

Head and neck cancer remains a morbid and often fatal disease and at the present time few effective molecular markers have been identified. The purpose of the present work was to identify new molecular markers for head and neck squamous cell carcinoma (HNSCC). We applied methylation-sensitive arbitrarily primed PCR (MS/APPCR) to isolate sequences differentially methylated in HNSCC. The most frequently hypermethylated fragment we found maps close to a cytosine guanine dinucleotide (CpG) island on chromosome 9q33.2, and hypermethylation of this CpG island was associated with transcriptional silencing of an alternative transcript of the LHX6 gene. Using combined bisulfite restriction analysis (COBRA), hypermethylation of this fragment was detected in 13 of 14 (92.8%) HNSCC cell lines studied and 21 of 32 (65.6%) primary tumors, whereas little or no methylation was seen in 10 normal oral mucosa samples. We extended this investigation to other cancer cell lines and methylation was found in those derived from colon, breast, leukemia and lung, and methylation was also found in 12/14 primary colon tumors. These findings suggest that differentially methylated (DIME)-6 hypermethylation is a good cancer marker in HNSCC as well as in other kinds of neoplasias and confirm the importance of searching for markers of epigenetic dysregulation in cancer.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression analysis reveals novel altered genes in nasal polyps

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)