Flavipin from fungi as a potential inhibitor of rheumatoid arthritis signaling molecules.

Flavipin, a fungal lower molecular weight biomolecule (MW 196.16 g/mol), has not been yet extensively studied for beneficial preclinical and clinical applications. In recent years, various preclinical mouse models including adjuvant-induced arthritis (AIA) were employed to understand mechanisms associated with Rheumatoid arthritis (RA) and to develop new therapeutic drugs. In the current study, we studied the inhibitory effect of Flavipin on major signaling molecules involved in the inflammatory response during RA using both in-silico virtual interaction and in vivo mouse model of AIA. Our in-silico results clarified that Flavipin interacts with the tumor necrosis factor alpha (TNF-α) through conventional hydrogen binding (H-H) at one of TNF-α critical amino acids tyrosine residues, Tyr119, with binding energy (b.e.) -5.9. In addition, Flavipin binds to ATP-binging sites of the Jesus kinases, JAK1, JAK2 and JAK3, through H-H (b. e. between -5.8 and -6.1) and then it may inhibit JAKs, regulators of RA signaling molecules. Moreover, our molecular dynamics stimulation for the docked TNF-α/Flavipin complex confirmed the specificity and the stability of the interaction. In vitro, Flavipin is not toxic to normal cells at doses below 50 µM (its IC50 in normal fibroblast cell line was above 100 µM). However, in vivo, the arthritis score and hind paw oedema parameters were modulated in Flavipin treated mice. Consistent with the in-silico results the levels of the TNF-α, the nuclear transcription factor kappaB (NF-κB) and the signal transduction and activator of transcription (STAT3, downstream of JAKs) were modulated at joint tissues of the hind-paw of Flavipin/AIA treated mice. Our data suggest Flavipin as a potential therapeutic agent for arthritis can inhibit RA major signaling molecules.

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

OPA-Based Bifunctional Linker for Protein Labeling and Profiling.

Lysine residues have been considered as a routine conjugating site for protein chemical labeling and modification. The commercially available lysine-labeling agents have several limitations in labeling efficiency, stability, and cost. To pursue alternative protein lysine-labeling strategies, herein, we report the development of an ortho-phthalaldehyde (OPA)-based bifunctional linker suitable for protein chemical labeling and profiling. Among three designed OPA-based bifunctional linkers, OPA-NH-alkyne 5 was proved to be optimal for protein labeling with minimal protein turbidity. We further demonstrated OPA-NH-alkyne 5 was applicable for immediate capture of protein or proteome chemical labeling.

Anticancer potential of NF-κB targeting apoptotic molecule "flavipin" isolated from endophytic Chaetomium globosum.

BACKGROUND: Anticancer compounds from natural sources have drawn attention due to their structural diversity and relatively lesser side effects. Endophytic fungi are one such natural resource from, which plethoras of anticancerous compounds have been isolated. PURPOSE: The objective of the study was to isolate and characterize the bioactive metabolite from Chaetomium globosum that exhibits astonishing antiproliferative activity against cancerous cell lines. METHODS: Flavipin was isolated by bioassay-guided fractionation and identified using FT-IR, EI-MS and NMR studies. MTT assay was used to determine the cytotoxicity. Fluorescent staining (AO/EB) and DNA fragmentation studies confirmed the occurrence of apoptosis. Real time PCR and Western blotting were used to analyze the expression of apoptosis related genes and its proteins, respectively. RESULTS: Flavipin inhibited proliferation of A549, HT-29 and MCF-7 cancer cells in dose dependent manner with an IC50 concentration of 9.89 µg/ml, 18 µg/ml and 54 µg/ml, respectively, whereas it was comparatively less sensitive (IC50 = 78.89 µg/ml) against normal cell line (CCD-18Co). At IC50 concentration cancerous cells exhibited cell shrinkage and fragmentation of DNA, which indicated that flavipin induced apoptotic cell death. In treated cells there is an up-regulation of p53 gene and its associated protein, whereas reciprocal expression was observed in BCL-2 gene and its protein. Furthermore, western blotting results also showed down-regulation of NFκB. CONCLUSION: This is the first report on the antiproliferative activity of flavipin isolated from endophytic C. globosum and also proposed that interaction of flavipin with NFкB could be a possible mechanism for this activity. Flavipin induced apoptosis at low concentrations in cancer cell lines (A549, HT-29) and exhibited itself as a potential anticancer agent.

Design and synthesis of ortho-phthalaldehyde phosphoramidite for single-step, rapid, efficient and chemoselective coupling of DNA with proteins under physiological conditions.

ortho-Phthalaldehyde (OPA) phosphoramidite with high reaction activity was designed and synthesized for labelling oligodeoxynucleotides (DNA). The DNA modified with OPA (OPA-DNA) can covalently couple with native proteins rapidly and efficiently via a condensation reaction with the formation of phthalimidines, which provides a highly efficient method for bioconjugation of DNA and native proteins under physiological conditions.

Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells.

BACKGROUND: Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility. METHODS: For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot. RESULTS: The in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4). CONCLUSION: Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients.

Flavipin in Chaetomium globosum CDW7, an endophytic fungus from Ginkgo biloba, contributes to antioxidant activity.

1,2-Benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl (flavipin) was found to be antagonistic against nematodes and fungi. Here we demonstrated that flavipin is a potent antioxidant in vitro and in vivo, which has great potential in the therapy for free radical-associated diseases. Therefore, flavipin-producing bio-source was screened from 80 endophytes in Ginkgo biloba. Seven endophytic fungi were able to synthesize antioxidant substances and identified by ITS rDNA sequences. Among them, Chaetomium globosum CDW7 was a remarkable producer of flavipin. The fermentation parameters of CDW7 were then optimized for high flavipin production. Cultured under the optimal condition (25 °C, 100/250 mL flask, 12 discs/flask, 150 rpm, pH 6.5) for 14 days, CDW7 was able to synthesize flavipin at a production of 315.5 mg/L. In addition, flavipin output was positively correlated to antioxidant activities of crude extracts with a correlation coefficient of 0.8235, indicating that flavipin was the major antioxidant component of CDW7's metabolites. These data demonstrated that CDW7 was a highly yielded bio-source of antioxidant flavipin.

Determination of sphingoid bases from hydrolyzed glucosylceramide in rice and wheat by online post-column high-performance liquid chromatography with O-phthalaldehyde derivatization.

We developed a determination method for sphingoid bases using online post-column high-performance liquid chromatography (HPLC) with O-phthalaldehyde (OPA) derivatization. Good separation was achieved using a reversed-phase column and eluting with 50% acetonitrile containing formic acid and heptafluorobutyric acid. Using these conditions, an excellent linearity (R² > 0.999) was achieved using standard solutions of sphinganine (d18:0), sphingosine (d18:1(4t)), 4-hydroxy-sphinganine (t18:0), glucosylsphingosine (glc-d18:1(4t)), and galactosylsphingosine (gal-d18:1(4t)). Plant glucosylceramides were hydrolyzed with 1 M aqueous HCl in methanol for 18 h at 90°C, followed by extraction of sphingoid bases with diethyl ether in preparation for analysis using the proposed HPLC conditions. The glc-d18:1(4t) standard was also hydrolyzed and analyzed by HPLC using the same procedure, and the d18:1(4t) peak obtained from the hydrolyzed glc-d18:1(4t) standard was used as a reference for calculation. We also confirmed the applicability of this method to the analysis of sphingoid bases in rice and wheat, obtaining relative standard deviations of 8.0% for rice and 4.6% for wheat. The recoveries of spiked rice and wheat samples were 104% and 106%, respectively. Our proposed method enables the straightforward determination of sphingoid bases without expensive facilities, employing fluorescence detection of OPA derivatives.

2-Formylcinnamaldehyde formation yield from the OH radical-initiated reaction of naphthalene: effect of NO(2) concentration.

Naphthalene, typically the most abundant polycyclic aromatic hydrocarbon in the atmosphere, reacts with OH radicals by addition to form OH-naphthalene adducts. These OH-naphthalene adducts react with O(2) and NO(2), with the two reactions being of equal importance in air at an NO(2) mixing ratio of ∼60 ppbv. 2-Formylcinnamaldehyde [o-HC(O)C(6)H(4)CH═CHCHO] is a major product of the OH radical-initiated reaction of naphthalene, with a yield from the reaction of OH-naphthalene adducts with NO(2) of ∼56%. We have measured, on a relative basis, the formation yield of 2-formylcinnamaldehyde from the OH radical-initiated reaction of naphthalene in air at average NO(2) concentrations of 1.2 × 10(11), 1.44 × 10(12), and 1.44 × 10(13) molecules cm(-3) (mixing ratios of 0.005, 0.06, and 0.6 ppmv, respectively). These NO(2) concentrations cover the range of conditions corresponding to the OH-naphthalene adducts reacting ∼90% of the time with O(2) to ∼90% of the time with NO(2). The 2-formylcinnamaldehyde formation yield decreased with decreasing NO(2) concentration, and a yield from the OH-naphthalene adducts + O(2) reaction of 14% is obtained based on a 56% yield from the OH-naphthalene adducts + NO(2) reaction. Based on previous measurements of glyoxal and phthaldialdehyde from the naphthalene + OH reaction and literature data for the OH radical-initiated reactions of monocyclic aromatic hydrocarbons, the reactions of OH-naphthalene adducts with O(2) appear to differ significantly from the OH-monocyclic adduct + O(2) reactions.

Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase.

We report here a method of simultaneously quantifying glucosylceramide (GlcCer) and galactosylceramide (GalCer) by normal-phase HPLC using O-phtalaldehyde derivatives. Treatment with sphingolipid ceramide N-deacylase which converts the cerebrosides in the sample to their lyso-forms was followed by the quantitative labeling of free NH(2) groups of the lyso-cerebrosides with O-phtalaldehyde. Using this method, 14.1 pmol of GlcCer and 10.4 pmol of GalCer, and 108.1 pmol of GlcCer and 191.1 pmol of GalCer were detected in zebrafish embryos and RPMI 1864 cells, respectively, while 22.2 pmol of GlcCer but no GalCer was detected in CHOP cells using cell lysate containing 100 microg of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 microg of protein under the conditions used, which corresponds to approximately 10(3) to 10(5) RPMI cells and 5 to 80 zebrafish embryos. The present method clearly revealed that the treatment of RPMI cells with a GlcCer synthase inhibitor P4 resulted in a marked decrease in GlcCer but not GalCer, concomitantly with a significant decrease in the GlcCer synthase activity. On the other hand, GlcCer but not GalCer increased 2-fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, the treatment of CHOP cells with ciclosporin A increased GlcCer possibly due to the inhibition of LacCer synthase. A significant increase in levels of GlcCer in fibroblasts from patients with Gaucher disease was clearly shown by the method. The proposed method is useful for the determination of GlcCer and GalCer levels in various biological samples.

Formation and reactions of 2-formylcinnamaldehyde in the OH radical-initiated reaction of naphthalene.

2-Formylcinnamaldehyde [O-HC(O)C6H4CH=CHCHO] is a major product of the OH radical-initiated reaction of naphthalene, the atmospherically most abundant polycyclic aromatic hydrocarbon. Previous studies indicate that 2-formylcinnamaldehyde undergoes photolysis as well as reaction with OH radicals. We have used direct air sampling atmospheric pressure ionization mass spectrometry (API-MS) to monitor 2-formylcinnamaldehyde as its protonated molecular ion during OH radical-initiated reactions of naphthalene. From the time-dependent behavior of the 2-formylcinnamaldehyde signal, ratios of (2-formylcinnamaldehyde removal rate/naphthalene reaction rate) were determined over a range of approximately 3 in (OH radical concentration/ light intensity). With an estimated rate constant for the reaction of OH radicals with 2-formylcinnamaldehyde of 5.3 x 10(-11) cm3 molecule(-1) s(-1), the photolysis rate of 2-formylcinnamaldhyde by blacklamps was determined to be approximately equal to that of NO2. Photolysis of 2-formylcinnamaldehyde will be the dominant loss process in the atmosphere, with an estimated lifetime of 2-formylcinnamaldehyde of approximately 120 s at a solar zenith angle of 30 degrees. Our data were used to re-evaluate the previous 2-formylcinnamaldehyde measurements of Sasaki et al. (Environ. Sci. Technol. 1997, 31, 3173-3179) and derive a 2-formylcinnamaldehyde formation yield from the OH radical reaction of naphthalene in the presence of NO of 56(-10)(+15)%.

Difluoro analogue of UCS15A triggers activation of exogenously expressed c-Src in HCT 116 human colorectal carcinoma cells.

UCS 15A, an antibiotic produced by Streptomyces sp., has been reported to specifically disrupt SH3 domain-mediated interactions in eukaryotic cells. Interestingly, in the case of the non-receptor tyrosine kinase Src, UCS15A was effective in suppressing the SH3 domain-mediated intermolecular rather than intramolecular interactions, and thus prevented Src interactions with certain downstream effectors without affecting Src kinase activity. Here the synthesis of a novel difluoro analogue of UCS15A is described. The effects of this compound (8) on Src activity were tested in HCT 116 colorectal carcinoma cells engineered for inducible expression of c-Src. The presence of compound (8) resulted in the increased activity of the induced c-Src implicating that (8) acts as a c-Src activator in vivo. These observations are supported by computer modelling studies which suggest that the aldehyde group of (8) may covalently bind to a lysine residue in the SH2-kinase linker region situated in the proximity of the SH3 domain, which could promote a conformational change resulting in increased Src activity.

Determination of fumonisins B1 and B2 in Portuguese maize and maize-based samples by HPLC with fluorescence detection.

Fumonisins B(1) (FB1) and fumonisin B2 (FB2) are the main members of a family of mycotoxins produced by Fusarium verticillioides, Fusarium proliferatum, and other fungi species of the section Liseola. The present work shows the results of comparative studies using two different procedures for the analysis of fumonisins in maize and maize-based samples. The studied analytical methods involve extraction with methanol/water, dilution with PBS, and clean-up through immunoaffinity columns. Two reagents (o-phthaldialdehyde and naphthalene-2,3-dicarboxaldehyde) were studied for formation of fluorescent derivatives. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of fumonisins in maize involved extraction with methanol/water (80:20), clean-up with an immunoaffinity column, and derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). The limit of detection was 20 microg kg(-1) for FB1 and 15 microg kg(-1) for FB2. Recoveries of FB1 and FB2 ranged from 79% to 99.6% for maize fortified at 150 microg kg(-1) and 200 microg kg(-1), respectively, with within-day RSDs of 3.0 and 2.7%. The proposed method was applied to 31 samples, and the presence of fumonisins was found in 14 samples at concentrations ranging from 113 to 2,026 microg kg(-1). The estimated daily intake of fumonisins was 0.14 microg kg(-1) body weight per day.

Behavior and characteristics of the o-phthaldialdehyde derivatives of n-C6-C8 amines and phenylethylamines with four additive SH-containing reagents.

The stability and characteristics of the C6-C8 n-aliphatic and phenylethylamines have been investigated as their o-phthaldialdehyde (OPA)/3-mercaptopropionic acid, OPA/N-acetyl-L-cysteine, OPA/2-mercaptoethanol and OPA/ethanethiol derivatives. Stoichiometric studies have been followed by photodiode array and fluorescence detection, simultaneously, while the composition of derivatives was confirmed by on line HPLC-electrospray ionization (ESI)-MS measurements. All four amines having in their original structure the NH2-CH2- moiety in accordance with the C1-C4 aliphatic, mono and diamines and amino acids of the same structure--furnished more than one OPA derivative: their initially formed isoindoles transform to further ones. Depending on the composition of the OPA reagents and on the pH of derivatizations different type of transformed species have been identified, in various proportions. Applying the OPA/SH additive reagent in the molar ratio of 1/3, favors the formation of one additional OPA molecule-containing isoindole, while using the OPA/SH additive (1/50) reagent resulted in the formation of one additional SH additive-containing species, identified and measured at the first time by HPLC. Transformation rate and stability of derivatives proved to be associated with the composition of the OPA reagent, with the type of the SH additive, with the pH of derivatizations, and, in selected cases also with the chain length of the amine. Results of stoichiometric and mechanism studies have been utilized to define optimum analytical conditions.

o-Phthaldialdehyde derivatization of histidine: stoichiometry, stability and reaction mechanism.

The irregular behavior of histidine in its reaction with the o-phthaldialdehyde (OPA) reagents has been studied. Histidine provides more than one OPA derivative. Similarly to all those primary amino group-containing compounds that do have in their initial structure the -CH2-NH2 moiety. The ratio of histidine's initially formed and transformed OPA derivatives depends on the temperature: very likely due to the fact that elevated temperature favors the intra-molecular rearrangement of histidine resulting in the formation of the -CH2-NH2 moiety-containing tautomer(s). The higher the temperature the higher the amount of the transformed species. The composition of the initially and transformed OPA derivatives of histidine were identified on the basis of their on-line HPLC-electrospray ionization (ESI) MS spectra and computations. The initially formed species has been identified as the classical isoindole, while the transformed one contains an additional OPA molecule.

Analysis of 2-difluoromethyl-DL-ornithine in human plasma, cerebrospinal fluid and urine by cation-exchange high-performance liquid chromatography.

An analytical method has been developed based on cation-exchange liquid chromatography for the measurement of 2-difluoromethyl-DT-ornithine (DFMO) in human plasma, cerebrospinal fluid (CSF) and urine. Fluorescence detection at excitation/emission wavelengths of 340/440 nm is followed by postcolumn derivatization with o-phthalaldehyde-2,mercaptoethanol. All calibration ranges yielded linear relationships with correlation coefficients better than 0.999. In each case the limit of quantitation was equal to the lowest value of the standard curve. The variability of the assay, expressed as relative standard deviations, was less than 7.1%, 15.3% and 7.1% for plasma, CSF and urine, respectively. The accuracy of the assay (expressed as relative errors) ranged between 4.3% and 2.0% for plasma analysis, between -0.1% and 14.0% for CSF analysis and between -8.0% and 2.0% for urine analysis. Plasma, CSF and urinary DFMO concentrations were measured in samples obtained from patients undergoing treatment for trypanosomiasis. The method was found to be applicable for the measurement of DFMO levels in human body fluids for the determination of pharmacokinetic parameters in clinical studies.

Biochemical and cytotoxic actions of 3,6-dihydroxy-4,5-dimethylphthalaldehyde in sarcoma 180 cells.

The replication of Sarcoma 180 cells in culture was inhibited by 3,6-dihydroxy-4,5-dimethylphthalaldehyde (HMPA). The inhibition of growth caused by HMPA was evident after treatment of cells with drug for only 15 min. This exposure period caused decreased in (a) cloning efficiency, (b) transport and/or phosphorylation of [3H]thymidine and [3H]uridine, (c) incorporation of radioactive nucleosides into acid-insoluble material, and (d) incorporation of [3H]leucine into protein. Examination of the cytotoxicities of the model compounds 2,3,5,6-tetramethyl-1,4-dihydroquinone (durohydroquinone) and o-phthalaldehyde indicated that the dialdehyde portion of the molecule was responsible for the cytocidal effects of HMPA. The ratio of adenosine triphosphate to adenosine diphosphate in the acid-soluble fraction of Sarcoma 180 cells incubated in vitro with HMPA for 45 min was reduced in a concentration-dependent manner. The reduction in the ATP pool size produced by HMPA contrasts with the action of the periodate oxidation product of cytidine dialdehyde, which has been reported to increase the intracellular concentration of adenosine triphosphate.