Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

Occurrence of fumonisins (B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>3</Subscript>) in maize-based food and feed samples from Indonesia

A survey to evaluate the contamination level of total fumonisins in maize-based foodstuffs, maize and feed from Indonesia is described. The analyses were carried out by enzyme-linked immunosorbent assay (ELISA). Samples were collected from local retail stores around Yogyakarta, Indonesia between February and May 2001. The 101 samples were classified into six categories, i.e. industrially-produced food (n=24), products of small food manufacturers (n=17), maize flour (n=4), maize for food (n=9), maize for feed (n17), and formulated feed (n30). Control of the method showed that the detection limit was 8.7 μg/kg and repeatability is shown by relative standard deviation (RSD) of analyses of contaminated maize (n=5) of 10 %. Results of analyses indicate that 80 samples analysed were contaminated over a large range from 10.0-3307 pg/kg, and the concentration of fumonisins depended on the type of sample. Of four samples of maize flour, none were contaminated (below detection limit). Of 24 samples of industrially produced food, 14 were contaminated in the range 22.8 - 105 μg/kg and 18 of 19 food samples from small manufacturers were contaminated ranging from 12.9 to 234 μg/kg. The highest contamination was observed in maize samples: six of ten samples of maize for food were contaminated between 68.0 - 2471 μg/kg and 16 of 17 samples for feed contained fumonisins over a large range from 17.6 to 3306 μg/kg.     

Increased Vascular Resistance by Prostaglandins B<Subscript>1</Subscript> and B<Subscript>2</Subscript> in the Isolated Rat Pancreas

IN view of the possibility that prostaglandins (PG) regulate local blood flow¹, we are investigating this activity in the pancreas. We have already found that PGE₂ reduces vascular resistance in the perfused rat pancreas whereas PGF_2α has the opposite effect². These effects were seen at low doses (0.1 µg/ml.) and with good reproducibility.     

Noble gas supported boron-pentagonal clusters B<Subscript>5</Subscript>Ng<Subscript>n</Subscript><Superscript>3+</Superscript>: exploring the structures and bonding

A novel type of trivalent BNg five-membered cational species B₅Ng_n³⁺(Ng = He~Rn, n = 1~5) has been found and investigated theoretically using the B3LYP and MP2 methods with the def2-QZVPPD and def2-TZVPPD basis sets. The geometry, harmonic vibrational frequencies, bond energies, charge distribution, bond nature, aromaticity, and energy decomposition analysis of these structures were reported. The calculated B?Ng bond energy is quite large (the averaged bond energy is in the range of 209.2~585.76 kJ mol^-1) for heavy rare gases and increases with the Ng atomic number. The analyses of the molecular wavefunction show that in the BNg compounds of heavy Ng atoms Ar~Rn, the B?Ng bonds are of typical covalent character. Nuclear independent chemical shifts display that both B₅³⁺ and B₅Ng_n³⁺(n=1~5) have obvious aromaticity. Energy decomposition analysis shows that these BNg compounds are mainly stabilized by the σ-donation from the Ng valence p orbital to the B₅³⁺ LUMO. These findings offer valuable clues toward the design and synthesis of new stable Ng-containing compounds.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

Effects of vitamin B<Subscript>12</Subscript> on murine embryonic dexamethasone-induced cleft palates using <Superscript>1</Superscript>H-NMR-based metabonomic analysis

Metabonomics using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and multivariate data analysis of blood plasma samples can be used to characterize metabolic differences between healthy and diseased organisms, which can reveal important information about the causes of the disease. Here we evaluated whether the ¹H-NMR-based metabonomic method can detect differences in blood plasma between healthy pregnant mice (outbred C57BL/6J strain) and pregnant mice injected with dexamethasone (Dex) to induce cleft palate. Both groups were injected with vitamin B₁₂. We found some metabolic differences from the “outliers” among the mice, indicating that vitamin B₁₂ protected against Dex-induced Cleft lip with or without palate (CLP) formation.     

An antibody-based microarray assay for the simultaneous detection of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript>

Advances in microsystem technology have enabled protein and nucleic acid-based microarrays to be used in various applications, including the study of diseases, drug discovery, genetic screening, and clinical and food diagnostics. Analytical methods for the detection of mycotoxins, however, remain largely based on thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or enzyme-linked Immunosorbent assay (ELISA) . The aim of our work, therefore, was to transfer an immunological assay from microtitrr plates into microarray format, in order to develop a multiparametric, rapid, sensitive and inexpensive method for the detection of mycotoxins for use in food safety applications. Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests but also peptide, antigen, and antibody assays, using different formats of microarrays, have evolved within the last decade. Antibody-based microarrays provide a powerful tool that can be used to generate rapid and detailed expression profiles of a defined set of analytes in complex samples and are potentially useful for generating rapid immunological assays of food contaminants. In this paper, we report a feasibility study of the application of antibody microarrays for the simultaneous (or independent) detection of two common mycotoxins, Aflatoxin B₁ and Fumonisin B₁. We present the development of microarray detection of aflatoxin B1 and fumonisin B1 in standard solutions with detection limits of 3 ng/ml of AFB1 and 43 ng/ml for FB1, and have developed a competitive immunoassay in microarray format for simultaneous analyses. The quality of the microarray data is comparable to data generated by microplate-based immunoassay (ELISA), but further investigations are needed in order to characterise our method more fully. We hope that these preliminary results might suggest that further research is warranted in order to develop hapten microarrays for the immunochemical simultaneous analysis of mycotoxins, as well as for other small molecules (e.g. bacterial toxins or biological warfare agents).     

Einsatz einer kombinierten Immunoaffinitätssäule zum Nachweis von Aflatoxinen (B<Subscript>1</Subscript>, G<Subscript>1</Subscript>, B<Subscript>2</Subscript>, G<Subscript>2</Subscript>), Ochratoxin und Zearalenon

A method for the combined determination of the mycotoxins aflatoxin B₁, G₁, B₂, G₂, ochratoxin A and zearalenone in cereals and feed is described. After extraction with acetonitrile/water or methanol/water the cleaning takes place with new combined immunoaffinity clean-up column “AflaOchraZea” by VICAM. When the mycotoxins are determined in different cereals with this new type of clean-up column low detection limits and high recovery rates can be reached similar to those obtained by using separate immunoaffinity clean-up colums for the said mycotoxins.     

Fumonisin B<Subscript>1</Subscript>, a sphingoid toxin,is a potent inhibitor of the plasma membrane H<Superscript>+</Superscript>-ATPase

Fumonisin B₁ (FB₁) is an amphipathic toxin produced by the pathogenic fungus Fusarium verticillioides which causes stem, root and ear rot in maize (Zea mays L.). In this work, we studied the action of FB₁ on the plasma membrane H⁺-ATPase (EC 3.6.1.34) from germinating maize embryos, and on the fluidity and lipid peroxidation of these membranes. In maize embryos the toxin at 40 M inhibited root elongation by 50% and at 30 M decreased medium acidification by about 80%. Irrespective of the presence and absence of FB₁, the H⁺-ATPase in plasma membrane vesicles exhibited non-hyperbolic saturation kinetics by ATPH-Mg, with Hill number of 0.67. Initial velocity studies revealed that FB₁ is a total uncompetitive inhibitor of this enzyme with an inhibition constant value of 17.5±1 M. Thus FB₁ decreased V_max and increased the apparent affinity of the enzyme for ATP-Mg to the same extent. Although FB₁ increased the fluidity at the hydrophobic region of the membrane, no correlation was found with its effect on enzyme activity, since both effects showed different FB₁-concentration dependence. Peroxidation of membrane lipids was not affected by the toxin. Our results suggest that, under in vivo conditions, the plasma membrane H⁺-ATPase is a potentially important target of the toxin, as it is inhibited not only by FB₁ but also by its structural analogs, the sphingoid intermediates, which accumulate upon the inhibition of sphinganine N-acyltransferase by this toxin.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Theoretical study of the novel sandwich compound [Au<Subscript>3</Subscript>Cl<Subscript>3</Subscript>Tr<Subscript>2</Subscript>]<Superscript>2+</Superscript>

A theoretical study of a sandwich compound with a metal monolayer sheet between two aromatic ligands is presented. A full geometry optimization of the [Au₃Cl₃Tr₂]²⁺ (1) compound, which is a triangular gold(I) monolayer sheet capped by chlorines and bounded to two cycloheptatrienyl (Tr) ligands was carried out using perturbation theory at the MP2 computational level and DFT. Compound (1) is in agreement with the 18–electron rule, the bonding nature in the complex may be interpreted from the donation interaction coming from the Tr rings to the Au array, and from the back-donation from the latter to the former. NICS calculations show a strong aromatic character in the gold monolayer sheet and Tr ligands; calculations done with HOMA, also report the same aromatic behavior on the cycloheptatrienyl fragments giving us an insight on the stability of (1). The Au –Au bond lengths indicate that an intramolecular aurophilic interaction among the Au(I) cations plays an important role in the bonding of the central metal sheet. Figure (a) Ground state geometry of complex 1; (b) Top view of compound 1 and Wiberg bond orders computed with the MP2/B1 computational method; (c) Lateral view of compound 1 and NICS values calculated with the MP2/B1 method; the values in parenthesis were obtained at the VWN/TZP level     

Aflatoxin B<Subscript>1</Subscript> levels in groundnut products from local markets in Zambia

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B₁ (AFB₁) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB₁ contamination levels in both types of groundnut products with maximum AFB₁ levels of 11,100 μg/kg (groundnut kernels) and 3000 μg/kg (milled groundnut powder). However, paired t test analysis showed that AFB₁ contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB₁ levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB₁ were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

Aflatoxins B<Subscript>1</Subscript> and M<Subscript>1</Subscript>: risks related to milk produced in Brazil

This work shows data on the occurrence of aflatoxins in milk produced in Brazil. A review of the literature on this contamination. Several studies carried out in Brazil show that levels of aflatoxin M₁ in milk are higher than the ones established by the legislation, an evidence of the lack of control and inspection of these mycotoxins. Taking into account that milk has been widely consumed as an important source of nutrients, mainly by children, it is fundamental to carry out a thorough study of the occurrence of aflatoxins and take measures to mitigate milk contamination.     

Production of fumonisins B<Subscript>2</Subscript> and B<Subscript>4</Subscript> in <Emphasis Type="Italic">Tolypocladium</Emphasis> species

Tolypocladium inflatum is known primarily for its production of the cyclosporines that are used as an immunosuppressive drug. However, we report here the production of the carcinogenic fumonisins B₂ and B₄ by this biotechnologically relevant fungal genus. These mycotoxins were detected in 11 strains tested from three species: Tolypocladium inflatum, T. cylindrosporum, and T. geodes. Production of fumonisins by Fusarium spp. and Aspergillus niger is highly medium- and temperature-dependent, so the effect of these parameters on fumonisin production by three T. inflatum strains was studied. Maximum production was achieved on media with high sugar content incubated at 25–30°C. Since these results demonstrate that fumonisin production could be widespread within the genus Tolypocladium, the potential contamination of commercial cyclosporine preparations with fumonisins needs to be investigated.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Solvent-dependent transformation of aflatoxin B<Subscript>1</Subscript> in soil

To date, all studies of aflatoxin B₁ (AFB₁) transformation in soil or in purified mineral systems have identified aflatoxins B₂ (AFB₂) and G₂ (AFG₂) as the primary transformation products. However, identification in these studies was made using thin layer chromatography which has relatively low resolution, and these studies did not identify a viable mechanism by which such transformations would occur. Further, the use of methanol as the solvent delivery vehicle in these studies may have contributed to formation of artifactual transformation products. In this study, we investigated the role of the solvent vehicle in the transformation of AFB₁ in soil. To do this, we spiked soils with AFB₁ dissolved in water (93:7, water/methanol) or methanol and used HPLC-UV and HPLC-MS to identify the transformation products. Contrasting previous published reports, we did not detect AFB₂ or AFG₂. In an aqueous-soil environment, we identified aflatoxin B_2a (AFB_2a) as the single major transformation product. We propose that AFB_2a is formed from hydrolysis of AFB₁ with the soil acting as an acid catalyst. Alternatively, when methanol was used, we identified methoxy aflatoxin species likely formed via acid-catalyzed addition of methanol to AFB₁. These results suggest that where soil moisture is adequate, AFB₁ is hydrolyzed to AFB_2a and that reactive organic solvents should be avoided when replicating natural conditions to study the fate of AFB₁ in soil.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

BODIPY for photodynamic therapy applications: computational study of the effect of bromine substitution on <Superscript>1</Superscript>O<Subscript>2</Subscript> photosensitization

Density functional theory and its time-dependent extension (DFT, TDDFT) were employed to establish the feasibility of using a series of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) in photodynamic therapy. Their absorption electronic spectra, singlet–triplet energy gaps, and spin–orbit matrix elements were computed and are discussed here. The effects of bromine substitution on the photophysical properties of BODIPY were elucidated. The investigated compounds were found to possess different excited triplet states that lie below the energy of the bright excited singlet state (S₁ or S₂), depending on the positions occupied by the bromine atoms. The computed spin–orbit matrix elements for the radiationless intersystem crossing S_n?→ ?T_m and the relative singlet–triplet energy gaps allowed the prediction of plausible nonradiative decay pathways for the production of singlet excited molecular oxygen, the key cytotoxic agent in photodynamic therapy.

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Graphical Abstract The photophysical properties affected by the presence of bromine atoms in different positions of a BODIPY core have been here elucidated. In particular it has been found that SOC values strongly depend on the position of heavy atoms into the BODIPY core, suggesting positions 1 and 7 as the best ones to enhance the ISC kinetics

     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.