Cellular autofluorescence is magnetic field sensitive.

We demonstrate, by direct, single-cell imaging kinetic measurements, that endogenous autofluorescence in HeLa cells is sensitive to the application of external magnetic fields of 25 mT and less. We provide spectroscopic and mechanistic evidence that our findings can be explained in terms of magnetic field effects on photoinduced electron transfer reactions to flavins, through the radical pair mechanism. The observed magnetic field dependence is consistent with a triplet-born radical pair and a B1/2 value of 18.0 mT with a saturation value of 3.7%.

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

Characterization of ankaflavin from Penicillium aculeatum and its cytotoxic properties.

The pigment was extracted from Penicillium aculeatum, purified and characterized as Ankaflavin by spectroscopic analysis. The stability of the pigment was determined under various conditions and was found to possess high stability. The cytotoxicity property of the purified pigment was determined by MTT assay in MCF-7, HCT116 and PC-3 and the studies were compared with its activity in CHOK1 cells. In MCF-7 and in CHOK 1 cells, the pigment exhibited very less toxicity. However, significant cytotoxicity was observed in HCT116 and PC-3 cells with IC50 of 162 µg mL-1 and 85 µg mL-1 for HCT116 and PC-3 cells respectively. In vitro toxicity was tested by haemolysis assay and MTT assay in HEK 293 cells. The pigment showed least cytotoxicity (<5%) at 160 and 320 µg mL-1 concentrations HEK 293 cells and negligible (<5%) toxicity on human erythrocytes at 160 and 320 µg mL-1, the highest concentrations tested.

Inhibitory Effects of Streptomyces sp. MBTH32 Metabolites on Sortase A and Sortase A-Mediated Cell Clumping of Staphylococcus aureus to Fibrinogen.

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.

Secondary metabolites from the fermented rice of the fungus Monascus purpureus and their bioactivities.

Phytochemical investigation of the EtOAc-soluble fraction of the ethanolic extract of a yellow mutant of the fungus Monascus purpureus BCRC 38110 (Eurotiaceae) grown on rice resulted in the isolation of one new azaphilone derivative, monapurpureusone (1), one acetophenone metabolite isolated for the first time from natural source, monapurpureusin (2), along with four known compounds, TW94a (3), ergosterol (4), monascin (5), and ankaflavin (6). The structures and relative configurations of these compounds were elucidated by spectroscopic analyses, including 1D- and 2D-NMR spectroscopy and mass spectrometry, and by the comparison of their NMR data with those of related compounds. Some phytochemicals were evaluated for both anti-inflammatory activity through the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine-derived macrophages RAW264.7 cell lines and antioxidant activities.

Three new amino acid derivatives from edible mushroom Pleurotus ostreatus.

Three new amino acid derivatives, oxalamido-L-phenylalanine methyl ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome hydrolyzate (3), together with nine known compounds (4-12), were isolated from the solid culture of edible mushroom Pleurotus ostreatus. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute configurations of 1 and 2 were established by the chiral synthesis and confirmed by circular dichroism (CD) analysis of their total synthesis products and natural isolates. All new compounds were evaluated for their antioxidant effects, antimicrobial activities, and cytotoxic activity. Compounds 1-3 showed weak antifungal activities against Candida albicans with minimum inhibitory concentration (MIC) value of 500 µg/ml.

Isolation and characterization of 2-pyridone alkaloids and alloxazines from Beauveria bassiana.

Two novel compounds bearing heterocyclic nitrogen, 2-pyridone alkaloid (1) and alloxazine derivative (2), along with the known pretenellin B (3), pyridovericin (4) and lumichrome (5) were isolated from a culture of the entomopathogenic fungal strain Beauveria bassiana. The chemical structures of 2-pyridone alkaloid and alloxazine derivative were established on the basis of the interpretation of spectroscopic data. The isolated compounds were evaluated in a panel of five cancer cell lines and pyridovericin exhibited cytotoxicity (IC50, µM) against cancer cell lines: HL-60 (25.9 ± 0.3), HCT8 (34.6 ± 3.6), MDA-MB435 (34.8 ± 3.8) and SF295 (31.1 ± 0.6). Considering that other pyridone compounds display good cytotoxic activity, it would be suggested to obtain new semi synthetic derivatives of pyridovericin, for the development of new cytotoxic chemical entities.

Efficient production of lumichrome by Microbacterium sp. strain TPU 3598.

Lumichrome is a photodegradation product of riboflavin and is available as a photosensitizer and fluorescent dye. To develop new efficient methods of lumichrome production, we isolated bacterial strains with high lumichrome productivity from soil. The strain with highest productivity was identified as Microbacterium sp. strain TPU 3598. Since this strain inductively produced lumichrome when cultivated with riboflavin, we developed two different methods, a cultivation method and a resting cell method, for the production of large amounts of lumichrome using the strain. In the cultivation method, 2.4 g (9.9 mmol) of lumichrome was produced from 3.8 g (10.1 mmol) of riboflavin at the 500-ml scale (98% yield). The strain also produced 4.7 g (19.4 mmol) of lumichrome from 7.6 g (20.2 mmol) of riboflavin (96% yield) by addition of riboflavin during cultivation at the 500-ml scale. In the resting cell method, 20 g of cells (wet weight) in 100 ml of potassium phosphate buffer, pH 7.0, produced 2.4 g of lumichrome from 3.8 g of riboflavin (98% yield). Since the lumichrome production by these methods was carried out in suspension, the resulting lumichrome was easily purified from the cultivation medium or reaction mixture by centrifugation and crystallization. Thus, the biochemical methods we describe here are a significant improvement in terms of simplicity and yield over the existing chemical, photolytic, and other biochemical methods of lumichrome production.

Treatment of metabolic syndrome with ankaflavin, a secondary metabolite isolated from the edible fungus Monascus spp.

Edible fungi of the Monascus species have been used as traditional Chinese medicine in eastern Asia for several centuries. Monascus-fermented products possess a number of functional secondary metabolites, including anti-inflammatory pigments (such as monascin and ankaflavin [AK]), monacolins, and dimerumic acid. These secondary metabolites have anti-inflammatory, anti-oxidative, and anti-tumor activities. We found that AK positively regulates several transcription factors associated with the prevention of metabolic syndrome and other diseases, including peroxisome proliferator-activated receptor (PPAR)-gamma, PPAR-alpha, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). AK reduced hyperglycemia and enhanced pancreatic function via PPAR-gamma activation and increased lipid metabolism due to PPAR-alpha activation. The compound also exerted antioxidant effects via activation of Nrf2. These results suggest that AK belongs to the class of selective peroxisome proliferator-activated receptor modulators (SPPARMs), which are associated with a good safety profile when used in patients suffering from metabolic syndrome. Together with our studies to determine how AK production can be increased during Monascus fermentation, these data demonstrate the great potential of AK as a nutraceutical or therapeutic agent.

Five new secondary metabolites from Monascus purpureus-fermented Hordeum vulgare and Sorghum bicolor.

Long grains of Hordeum vulgare and Sorghum bicolor were individually fermented with Monascus purpureus MTCC 369 under solid state fermentation. The aqueous extract of Monascus which fermented H. vulgare and S. bicolor was found to contain five different new metabolites. Silica gel column chromatography of the aqueous extract with a linear gradient of ethyl acetate, acetonitrile and carbon tetrachloride (v/v) yielded five new metabolites named benzopranyl capriate (9H-1-isoprenyl-benzopyran-5-isopropanoic acid-6-ol-6-n-decanoate), shorghumoic acid (n-octadec-8,11-dien-7α-ol-1-oic acid) and sorghumflavin A (2-n-butyloxo-6-ß-hydroxy-7-ß-isoprenyl ankaflavin) from Monascus-fermented S. bicolor, while hordeumflavin B (2-n-undecanyloxo-7-ß-isoprenyl ankaflavin) and vulgaredilone (2-dodecanyl-7-ß isopranyl monoscodilone) from Monascus-fermented H. vulgare.

Identification of a small-molecule inhibitor of the interaction between Survivin and Smac/DIABLO.

The protein Survivin is selectively overexpressed in a variety of cancers, but not in normal tissues. It has been reported to be involved in cell survival and cell division. However, the molecular mechanisms involved in its function are not clear, although several binding partner proteins have been proposed to date. Here, we report the identification of a novel small molecule Survivin antagonist, which disrupts the Survivin-Smac/DIABLO interaction in cells. In order to identify Survivin-directed antagonists, we developed a high-throughput screening system based on AlphaScreen technology, which allows the identification of small molecules with the ability to inhibit the interaction of Survivin with Smac/DIABLO or INCENP in vitro. We screened chemical libraries, generated in-house, using this system and identified a 5-deazaflavin analog (compound 1) as a hit compound that selectively inhibited the interaction of Survivin with Smac/DIABLO but not INCENP. In cultured cells, compound 1 abrogated the formation of the complex between Survivin and Smac/DIABLO. In addition, this compound was able to sensitize cultured cells to doxorubicin-mediated DNA damage stress and synergistically enhance apoptotic cell death. Thus, the small-molecule inhibitor described here may serve as a proof-of-principle agent for discriminating between the multiple functions of Survivin.

Secondary metabolites from the red mould rice of Monascus purpureus BCRC 38113.

One new tetralone, monaspurpurone (1), was isolated from the EtOH extract of a yellow mutant of the fungus Monascus purpureus BCRC 38113 (Eurotiaceae) grown on rice, along with five known compounds, ß-sitosteryl palmitate (2), ergosterol (3), ankaflavin (4), monascin (5) and p-nitrophenol (6). They were characterised on the basis of spectral analysis and comparison with literature data. All the isolates were also evaluated for the scavenging properties towards the DPPH in TLC autographic and spectroscopic assays.

(1)H and (13)C NMR assignments of eight nitrogen containing compounds from Nocardia alba sp.nov (YIM 30243(T)).

An unprecedented new natural product named nocarsin A (1), 5H-4a,6,7a-triazacyclopenta[cd]indene-5,7(6H)-dione (1), together with seven known compounds lumichrome (2), cyclo (L-Leu-L-Tyr) (3), cyclo (L-Ala-L-Ile) (4), cyclo (L-Ala-L-Leu) (5), cyclo (L-Val-L-Ala) (6), 5-methyluracil (7) and uracil (8), was isolated from Nocardia alba sp.nov (YIM 30243(T)), which was isolated from a soil sample collected from Yunnan Province, P. R. China. NMR techniques including COSY, HSQC, ROESY, and HMBC were used to elucidate the structures of these compounds. We report the unambiguous assignments of the (1)H and (13)C NMR spectra of the new compound nocarsin A (1).

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis.

Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.

Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins. The HSCCC run was carried out with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that pure theaflavin, theaflavins-3-gallate, theaflavins-3'-gallate and theaflavin-3,3'-digallate could be obtained from crude theaflavins sample and black tea. The structures of the isolated compounds were positively confirmed by (1)H NMR and (13)C NMR, MS analysis, HPLC data and TLC data. Meanwhile, catechins including epigallocatechin gallate, gallocatechin gallate, epicatechin gallate and epigallocatechin were isolated from the aqueous extract of green tea by using the same solvent system. This study developed a modified method combined with enrichment theaflavins method by using HSCCC for separation of four individual theaflavins, especially for better separation of theaflavins monogallates.

Purification and characterization of alcohol oxidase from Paecilomyces variotii isolated as a formaldehyde-resistant fungus.

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6-10. The enzyme was stable in the pH range of pH 5-10. The enzyme retained full activity after incubation at 50 degrees C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K (m) values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l(-1), respectively.

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites.

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.

Inhibition of phospholipase Cgamma1 and cancer cell proliferation by lignans and flavans from Machilus thunbergii.

Thirteen compounds were isolated from the CH2Cl2 fraction of Machilus thunbergii as phospholipase Cgamma1 (PLCgamma1) inhibitors. These compounds were identified as nine lignans, two neolignans, and two flavans by spectroscopic analysis. Of these, 5,7-di-O-methyl-3',4'-methylenated (-)-epicatechin (12) and 5,7,3'-tri-O-methyl (-)-epicatechin (13) have not been reported previously in this plant. In addition, seven compounds, machilin A (1), (-)-sesamin (3), machilin G (5), (+)-galbacin (9), licarin A (10), (-)-acuminatin (11) and compound 12 showed dose-dependent potent inhibitory activities against PLCgamma1 in vitro with IC50 values ranging from 8.8 to 26.0 microM. These lignans, neolignans, and flavans are presented as a new class of PLCgamma1 inhibitors. The brief study of the structure activity relationship of these compounds suggested that the benzene ring with the methylene dioxy group is responsible for the expression of inhibitory activities against PLCgamma1. Moreover, it is suggested that inhibition of PLCgamma1 may be an important mechanism for an antiproliferative effect on the human cancer cells. Therefore, these inhibitors may be utilized as cancer chemotherapeutic and chemopreventive agents.

Subsecond separation of cellular flavin coenzymes by microchip capillary electrophoresis with laser-induced fluorescence detection.

In this article, it was demonstrated that a subsecond separation of cellular metabolites such as riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) was achieved using microchip capillary electrophoresis with laser-induced fluorescence detection. The influences of crucial parameters that governed analysis time (e.g., channel length and electric field for separation) and separation resolution (e.g., sample size) were investigated, both in theoretical aspects and experimental practice. Quantitative analyses were performed that exhibited linear dynamic range of two orders of magnitude, with calculated detection limits of 34, 201, and 127 nM for RF, FAD, and FMN, respectively. To test the validity of the method, it was successfully applied to characterize several recombinant flavin-binding domains in a human neuronal nitric oxide synthase.

Sequences of polypeptide antibiotics stilboflavins, natural peptaibol libraries of the mold Stilbella flavipes.

From the culture broths of the mold Stilbella flavipes CBS 146.81, a mixture of polypeptides could be isolated by adsorption on XAD polystyrene resin and purified by Sephadex LH-20 chromatography. Using preparative thin-layer chromatography (TLC) three groups of peptides, named stilboflavins (SF) A, B, and C could be separated. Each of the groups showed microheterogeneity when investigated by high-performance liquid chromatography (HPLC). Employing on-line HPLC-electrospray ionization tandem mass spectrometry in the positive and negative ionization mode, together with gas chromatography-selected ion monitoring mass spectrometry, enantioselective GC and quantitative amino acid analysis, the sequences of stilboflavins A and B could be determined. Exchange of Glu in stilboflavins A peptides (acidic) against Gln in stilboflavins B peptides (neutral) is the rational for different polarity of the peptide groups and their separatability by TLC. Since SF A and B are bioactive N-acetylated 20-residue peptides with a high proportion of alpha-aminoisobutyric acid and C-terminal bonded amino alcohols (either leucinol, isoleucinol or valinol) the peptides belong to the group of peptaibol antibiotics.