Antimicrobial activity of rhodomyrtone isolated from Rhodomyrtus tomentosa (Aiton) Hassk

Abstract

Rhodomyrtone was isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk grown in Vietnam using chromatographic methods. Its chemical structure was confirmed by means of spectroscopic data analysis. The pH drop measurement, enzyme activity assays and fluorescence stain were used to examine rhodomyrtone anticaries activity. It was found that rhodomyrtone suppressed acid production by Streptococcus mutans, a major cariogenic agent in human by inhibiting enzyme activities responsible for acid production and tolerance, including membrane bound enzymes F-ATPase and phosphotransferase system (PTS), as well as glycolysis enzymes glyceraldehyphosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) in cytoplasm with the IC₅₀ values of 24?μM, 19?μM, 23?μM and 28?μM, respectively. Moreover, 50?μM rhodomyrtone reduced biofilm biomass formed by S. mutans up to 59% (p?<?0.05). Fluorescent images indicated that cells on the biofilms were significantly killed. Thus, rhodomyrtone is a new and potential anticaries agent against S. mutans.     

The Pyruvate Dehydrogenase Multienzyme Complex

The three enzymes pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase constitute the pyruvate dehydrogenase multienzyme complex of E. coli; in mammals the complex also contains a kinase and a phosphatase. Multienzyme complexes are structural, functional, and regulatory units enabling the organism to operate more economically than with single enzymes. The pyruvate dehydrogenase multienzyme complex may stand at the switch-point between energy metabolism and gluconeogenesis.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast

A fungal strain of Aspergillus sp. (BRF 030) was isolated from the sediments collected in the northeast coast of Brazil, and the cytotoxic activity of its secondary metabolites was investigated against HCT-116 tumour cell line. The cytotoxicity-guided fractionation of the extracts from this fungus cultured in potato-dextrose-sea water for 14 days at room temperature yielded the hetero-spirocyclic γ-lactams pseurotin A (1), pseurotin D (2) and pseurotin FD-838 (7), the alkaloids fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6), methylsulochrin (4) and bis(dethio)bis(methylthio)gliotoxin (3). Among them, fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6) were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6).     

The In Vitro Anti-Cancer Activities and Mechanisms of Action of 9-Methoxycanthin-6-one from Eurycoma longifolia in Selected Cancer Cell Lines

An alkaloid compound from the hairy root culture of Eurycoma longifolia has been isolated and characterised as 9-methoxycanthin-6-one. The aims of these studies were to investigate the in vitro anti-cancer activities of 9-methoxycanthin-6-one against ovarian cancer (A2780, SKOV-3), breast cancer (MCF-7), colorectal cancer (HT29), skin cancer (A375) and cervical cancer (HeLa) cell lines by using a Sulphorhodamine B assay, and to evaluate the mechanisms of action of 9-methoxycanthin-6-one via the Hoechst 33342 assay and proteomics approach. The results had shown that 9-methoxycanthin-6-one gave IC₅₀ values of 4.04 ± 0.36 µM, 5.80 ± 0.40 µM, 15.09 ± 0.99 µM, 3.79 ± 0.069 µM, 5.71 ± 0.20 µM and 4.30 ± 0.27 µM when tested in A2780, SKOV-3, MCF-7, HT-29, A375 and HeLa cell lines, respectively. It was found that 9-methoxycanthin-6-one induced apoptosis in a concentration dependent manner when analysed via the Hoechst 33342 assay. 9-methoxycanthine-6-one were found to affect the expressions of apoptotic-related proteins, that were proteins pyruvate kinase (PKM), annexin A2 (ANXA2), galectin 3 (LGAL3), heterogeneous nuclear ribonucleoprotein A1 (HNRNP1A1), peroxiredoxin 3 (PRDX3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the differential analysis of 2-DE profiles between treated and non-treated 9-methoxycanthine-6-one. Proteins such as acetyl-CoA acyltransferase 2 (ACAA2), aldehyde dehydrogenase 1 (ALDH1A1), capping protein (CAPG), eukaryotic translation elongation factor 1 (EEF1A1), malate dehydrogenase 2 (MDH2), purine nucleoside phosphorylase (PNP), and triosephosphate isomerase 1 (TPI1) were also identified to be associated with A2780 cell death induced by 9-methoxycanthine-6-one. These findings may provide a new insight on the mechanisms of action of 9-methoxycanthin-6-one in exerting its anti-cancer effects in vitro.     

Synthetic studies directed toward the pseurotins. Part II. Synthesis of related highly functionalized furan-3(2H)-ones

The synthesis of 2,2-bis(hydroxymethyl)-4-methyl-5-phenylfuran-3(2H)-one ( 9 ), 5-[(1S,2S,Z)-1,2-(ethylidenedioxy)hex-3-enyl]-2,2-bis(hydroxymethyl)-4-methylfuran-3(2H)-one ( 24 ), and 5-[(1S,2S,Z)-1,2-(ethylidenedioxy)hex-3-enyl]-2-(hydroxymethyl)-4-methylfuran-3(2H)-one ( 28 ), which represent more advanced, suitably functionalized intermediates for the synthesis of pseurotin A ( 1 ), a secondary metabolite of Pseudeurotium ovalis STOLK , is described.     

Elucidation of Pseurotin Biosynthetic Pathway Points to Trans‐Acting C‐Methyltransferase: Generation of Chemical Diversity

Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Systematic deletion of the pseurotin biosynthetic genes in A. fumigatus and in vivo and in vitro characterization of the tailoring enzymes to determine the biosynthetic intermediates, and the gene products responsible for the formation of each intermediate, are described. Thus, the main biosynthetic steps leading to the formation of pseurotin A from the predominant precursor, azaspirene, were elucidated. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase C‐methyltransferase bifunctional fusion protein PsoF which appears to methylate the nascent polyketide backbone carbon atom in trans.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Capillary electrophoresis-integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L-lactate dehydrogenase via hydrophobic interactions

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K_m values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.     

Enzyme electrodes with substrate and co-enzyme amplification

The enzyme couples horseradish peroxidase/glucose dehydrogenase, glucose oxidase/glucose dehydrogenase, and cytochrome b₂/lactate dehydrogenase are applied in enzyme electrodes. Based on amplification by the recyclization reactions catalyzed by these two-enzyme systems, NADH, NAD⁺, glucose, lactate and pyruvate, are determined with 8–40-fold increased sensitivity compared to the unamplified reactions. Detection limits are 1.0 × 10^?6 M NADH, 1.2 × 10^?6 M NAD⁺, 8 × 10^?7 M glucose, and 3 × 10^?7 M lactate or pyruvate.     

Synthetic Studies Directed toward the Pseurotins. Part I. Synthesis of Related Furan-3(2H)-ones

According to a general concept for the total synthesis of pseurotin A ( 1 ), a secondary metabolite of Pseudeurotium ovalis STOLK , 5-[(1S,2S,Z)-1,2-dihydroxyhex-3-enyl]-2,2,4-trimethylfuran-3(2H)-one ( 17 ) was prepared. It is a model substance for the substituted furan-3(2H)-one moiety of 1 . The aldol condensation of the aldehyde 26 , derived from D-glucose, and the enolate of ketone 29 served as key reaction.     

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

A non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.     

Pseurotin, a new metabolite of Pseudeurotium ovalis Stolk having an unusual hetero-spirocyclic system.

The structure and absolute configuration of pseurotin ( 1 ), a new metabolite, isolated from culture filtrates of Pseudeurotium ovalis STOLK (Ascomycetes), has been shown to be 2-[1′(S), 2′ (S)-dihydroxhex-3′-ene-yl]-3-methyl-8(S)-methoxy-8-benzoyl-9(R)-hydroxy-(5S)-1-oxa-7-aza-spiro[4.4]non-2-ene-4, 6-dione ( 1 ), by spectral data and chemical transformations, and by X-ray analysis of its dibromo derivative 2 [1].     

Non-natural Cofactor and Formate-Driven Reductive Carboxylation of Pyruvate

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.     

Immobilization of Enzymes on Polychlorotrifluoroethylene Particles Packed in Small Columns

Abstract

The use of polychlorotrifluoroethylene (PCTFE) particles packed in 5–10 cm × 0.4 cm ID columns for the immobilization of enzymes by hydrophobic adsorption is investigated. Enzyme binding capacity of PCTFE is about 0.2 mg/gm of polymer. PCTFE-immobilized lactate dehydrogenase, alcohol dehydrogenase, and urease-glutamate dehydrogenase are demonstrated to be useful for the determinations of pyruvate, ethanol, and urea, respectively. Immobilized enzyme lifetimes are generally about 1–2 months.     

Oxidative trans to cis Isomerization of Olefins in Polyketide Biosynthesis

Geometric isomerization can expand the scope of biological activities of natural products. The observed chemical diversity among the pseurotin‐type fungal secondary metabolites is in part generated by a trans to cis isomerization of an olefin. In vitro characterizations of pseurotin biosynthetic enzymes revealed that the glutathione S‐transferase PsoE requires participation of the bifunctional C‐methyltransferase/epoxidase PsoF to complete the trans to cis isomerization of the pathway intermediate presynerazol. The crystal structure of the PsoE/glutathione/presynerazol complex indicated stereospecific glutathione–presynerazol conjugate formation is the principal function of PsoE. Moreover, PsoF was identified to have an additional, unexpected oxidative isomerase activity, thus making it a trifunctional enzyme which is key to the complexity generation in pseurotin biosynthesis. Through the study, we identified a novel mechanism of accomplishing a seemingly simple trans to cis isomerization reaction.     

Synergic effect of triphenylphosphine oxide (TPPO) on the extraction of Hg(II), Se(IV), Co(II) and Mn(II) with fluorinated pyrazolone (HPMTFP)

Extraction of Hg(II), Se(IV), Mn(II) and Co(II) has been carried out with 1-phenyl-3-methyl-4-trifluoroacetyl-2-pyrazolin-5-one (HPMTFP) and triphenylphosphine oxide (TPPO) into chloroform from pH 1–10. Quantitative extraction of Hg(II), Mn(II) and Co(II) at pH 4 with equimolar 0.05M (PMTFP+TPPO) in chloroform was observed. Se(IV) remains unextracted at this pH range. The stoichiometric composition of the extracted complexes M(PMTFP)₂·nH₂O (M=Mn, Hg), M(PMTFP)₂·2TPPO (M=Mn and Co) and Co(PMTFP)₂·TPPO·H₂O at less than 0.1M TPPO has been established. The formation constantsK _m,0 andK _m,n and stability constants _m,n have been computed. The analytical method developed was applied to the IAEA standard reference material (SRM) potato fluor V-4 for the determination of these elements using NAA technique.     

Impact of high pyruvate concentration on kinetics of rabbit muscle lactate dehydrogenase

In order to evaluate the effectiveness of l-lactate dehydrogenase (LDH) from rabbit muscle as a regenerative catalyst of the biologically important cofactor nicotinamide adenine dinucleotide (NAD), the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Despite robust literature describing the intricate complexations, the mammalian rabbit muscle LDH lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters, specifically at high pyruvate concentrations. Product inhibition by l-lactate was observed to reduce activity at concentrations greater than 25 mM, while expected substrate inhibition by pyruvate was significant above 4.3 mM concentration. The combined effect of ternary and binary complexes of pyruvate and the coenzymes led to experimental rates as little as a third of expected activity. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression:

PTP1B Inhibitory Secondary Metabolites from an Antarctic Fungal Strain Acremonium sp. SF-7394

Chemical investigation of the Antarctic lichen-derived fungal strain Acremonium sp. SF-7394 yielded a new amphilectane-type diterpene, acrepseudoterin (1), and a new acorane-type sesquiterpene glycoside, isocordycepoloside A (2). In addition, three known fungal metabolites, (−)-ternatin (3), [D-Leu]-ternatin (4), and pseurotin A (5), were isolated from the EtOAc extract of the fungal strain. Their structures were mainly elucidated by analyzing their NMR and MS data. The absolute configuration of 1 was proposed by electronic circular dichroism calculations, and the absolute configuration of the sugar unit in 2 was determined by a chemical method. The inhibitory effects of the isolated compounds on protein tyrosine phosphatase 1B (PTP1B) were evaluated by enzymatic assays; results indicated that acrepseudoterin (1) and [D-Leu]-ternatin (4) dose-dependently inhibited the enzyme activity with IC₅₀ values of 22.8 ± 1.1 μM and 14.8 ± 0.3 μM, respectively. Moreover, compound 1 was identified as a competitive inhibitor of PTP1B.     

Design and synthesis of novel rofecoxib analogs as potential cyclooxygenase (COX‐2) inhibitors: Replacement of the methylsulfonyl pharmacophore by a sulfonylazide bioisostere

A group of rofecoxib analogs, having a sulfonylazide (SO₂N₃) substituent in place of the methanesulfonyl (SO₂CH₃) pharmacophore at the meta‐position viz 3‐(4‐methyl, 4‐methoxy, or 4‐ethoxyphenyl)‐4‐(3‐sulfonylazidophenyl)‐2(5H)furanone ( 7a‐c ) and para‐position viz 3‐phenyl‐4‐(4‐sulfonylazidophenyl)‐2(5H)furanone ( 7d ), 3‐(4‐fluoro, or 4‐chlorophenyl)‐4‐(4‐sulfonylazidophenyl)‐2(5H)furanone ( 7e‐f ) of the C–4 phenyl ring, and 4‐(1‐oxido‐4‐pyridyl)‐3‐phenyl‐2(5H)furanone ( 12 ) were designed and synthesized for evaluation as selective cyclooxygenase‐2 (COX‐2) inhibitors. In vitro COX‐1/COX‐2 enzyme inhibition studies showed that 3‐phenyl‐4‐(4‐sulfonylazidophenyl)‐2(5H)furanone ( 7d ) inhibited COX‐1 selectively (COX‐1 IC₅₀ = 0.6659 μM; COX‐2 IC₅₀ > 100 μM) and 3‐(4‐fluorophenyl)‐4‐(4‐sulfonylazidophenyl)‐2(5H)furanone ( 7e ) inhibited both enzymes (COX‐1 IC₅₀ = 0.8494 μM; COX‐2 IC₅₀ = 1.7661 μM). A molecular modeling study was performed where 3‐(4‐fluorophenyl)‐4‐(4‐sulfonylazidophenyl)‐2(5H)furanone ( 7e ) was docked in the active site of murine COX‐2 isozyme, which showed that the sulfonylazido group inserts deep into the 2°‐pocket of COX‐2 where it undergoes both H‐bonding (Gln¹⁹², Phe⁵¹⁸) and weak electrostatic (Arg⁵¹³) interactions.