Effect of red pigment on insulin fibril formation in vitro

The influence of red pigment isolated from yeast Saccharomyces cerevisiae and its low molecular weight derivate on insulin amyloid fibril formation in vitro was studied. The red pigment derivative, which presumably lacked phosphoribosyl moiety due to acid hydrolysis, retained the ability to inhibit fluorescence intensity (FI) of amyloid bound Thioflavine T. It was found that FI inhibition depended on the concentration of both pigment forms. Both forms were also able to compete with Thioflavine T for amyloid fibril binding. Electron microscopy revealed that fibrils reduced in size in the presence of red pigment.     

The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.     

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987¹, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory,and examples describing applications of the method are provided.     

Replacement of light by dibutyryl-CAMP and CAMP in betacyanin synthesis

The effect of adenosine 3′,5′-cyclic monophosphate and its N⁶,O^2′- dibutyryl derivative (Bu₂-CAMP) on betacyanin formation in etiolated Amaranthus paniculatus seedlings was investigated. Both substances can replace the action of light in the synthesis of these pigments, the formation of which is controlled by phytochrome. The specificity of this mimicry is underlined by the observations that sodium butyrate does not promote any betacyanin formation and that theophylline enhances the effect of Bu₂-AMP. Puromycin inhibits the induction of betacyanin synthesis by Bu₂-CAMP just as it does the light-induced pigment formation. These findings suggest that phytochrome exerts its controlling role in the synthesis of betacyanins through the agency of CAMP.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Enhancing the anti-biofilm activity of 5-aryl-2-aminoimidazoles through nature inspired dimerisation

The increased tolerance of biofilms against disinfectants and antibiotics has stimulated research into new methods of biofilm prevention and eradication. In our previous work, we have identified the 5-aryl-2-aminoimidazole core as a scaffold that demonstrates preventive activity against biofilm formation of a broad range of bacterial and fungal species. Inspired by the dimeric nature of natural 2-aminoimidazoles of the oroidin family, we investigated the potential of dimers of our decorated 5-aryl-2-aminoimidazoles as biofilm inhibitors. A synthetic approach towards 2-aminoimidazole dimers linked by an alkyl chain was developed and a total of 48 dimers were synthesized. The linkers were introduced at two different positions, the N1-position or the N2-position, and the linker length and the substitution of the 5-phenyl ring (H, F, Cl, Br) were varied. Although, no clear correlation between linker length and biofilm inhibition was observed, a strong increase in anti-biofilm activity for almost all N1,N1′-linked dimers was obtained, compared to the respective monomers against Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. The N2,N2′-linked dimers, having a H- or F-substitution, were also found to show a strong increase in anti-biofilm activity compared to the respective monomers against these three bacterial species and against Pseudomonas aeruginosa. In addition, the obtained growth measurements suggest a broad concentration range with specific biofilm inhibition and no effect on the planktonic growth against Salmonella Typhimurium and Pseudomonas aeruginosa.     

Isolation and Characterization of Sexual Spore Pigments from Aspergillus nidulans

The homothallic ascomycete Aspergillus nidulans produces two types of pigmented spores: conidia and ascospores. The synthesis and localization of the spore pigments is developmentally regulated and occurs in specialized cell types. On the basis of spectroscopic evidence, we propose that the major ascospore pigment of A. nidulans (ascoquinone A) is a novel dimeric hydroxylated anthraquinone. The structure of ascoquinone A, as well as a comparison to model compounds, suggests that it is the product of a polyketide synthase. Previous studies have revealed that the conidial pigments from A. nidulans and a related Aspergillus species (A. parasiticus) also appear to be produced via polymerization of polyketide precursors (D. W. Brown, F. M. Hauser, R. Tommasi, S. Corlett, and J. J. Salvo, Tetrahedron Lett. 34:419-422, 1993; M. E. Mayorga and W. E. Timberlake, Mol. Gen. Genet. 235:205-212, 1992). The structural similarity between the ascospore pigment and the toxic anthraquinone norsolorinic acid, the first stable intermediate in the aflatoxin pathway, suggests an evolutionary relationship between the respective polyketide synthase systems.     

Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a Ki of 0.34 nM. The structural attributes and the slow, tight binding characteristics of NAIR implicated this compound as a transition state or reactive intermediate analog. However, it is unclear what molecular features of NAIR contribute to the mimetic properties for either of the two proposed mechanisms of AIR carboxylase. In order to gain additional information regarding the mechanism for the potent inhibition of AIR carboxylase by NAIR, a series of heterocyclic analogs were prepared and evaluated. We find that all compounds are weaker inhibitors than NAIR and that CAIR analogs are not alternative substrates for the enzyme. Surprisingly, rather subtle changes in the structure of NAIR can lead to profound changes in binding affinity. Computational investigations of enzyme intermediates and these inhibitors reveal that NAIR displays an electrostatic potential surface similar to a proposed reaction intermediate. The result indicates that AIR carboxylase is likely sensitive to the electrostatic surface of reaction intermediates and thus compounds which mimic these surfaces should possess tight binding characteristics. Given the evolutionary relationship between AIR carboxylase and N⁵-CAIR mutase, we believe that this concept extends to the mutase enzyme as well. The implications of this hypothesis for the design of selective inhibitors of the N⁵-CAIR mutase are discussed.     

Melanin and novel melanin precursors from Aeromonas media

Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product.     

Inhibition of sterol biosynthesis and accumulation of 2,3-oxidosqualene in bramble cell suspension cultures treated with 2-aza-2,3-dihydro-squalene and 2-aza-2,3-dihydrosqualene-N-oxide

2-Aza-2,3-dihydrosqualene (1), 2-aza-2,3-dihydro-squalene-N-oxide (2) and derivatives are new compounds designed to inhibit the 2,3-oxidosqualene-cycloartenol (lanosterol) cyclase. The effects of these compounds were studied on sterol biosynthesis in suspension cultures of bramble cells. Both 1 and 2 inhibited the growth of cells with an IC₅₀ of 11 μM for 1 and 21 μM for 2. When the cells grown in the presence of the two drugs were analysed, accumulation of squalene and 2,3-oxidosqualene was observed but no significant decrease of the total sterol content per g of dry weight of cells was noticed. Pulse experiments with [2-¹⁴C]acetate on 15-day-old cells treated with 1 resulted in a strong decrease of the incorporation of radioactivity into the 4-desmethyl sterol fraction. An IC₅₀ of 7.5 μM was determined when the cells were preincubated for a period of two hr with 1 or 2. This inhibition was correlated with an accumulation of [¹⁴C]-2,3-oxidosqualene and of [¹⁴C]-squalene. No [¹⁴C]-2,3:22(23)-dioxidosqualene was detectable in these conditions. Derivatives of 1 and 2 or similar compounds were also assayed; N-lauryl-dimethylamino-N-oxide (LDAO) was shown to be particularly effective and produced accumulation of enormous amounts of [¹⁴C]-2,3-oxidosqualene. Compound 1 (but not 2 or LDAO) leads also to the accumulation of a red pigment identified as lycopene. Our work confirms studies with enzymatic systems in demonstrating that 1, 2 and LDAO inhibit the 2,3-oxidosqualene-cycloartenol cyclase and provides evidence that the squalene synthetase and the Δ⁸ → Δ⁷-sterol isomerase are also inhibited.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Brilliant skyblue pigment formation from gardenia fruits

It is well known that blue pigment is formed by the reaction of amino acids with genipin, the hydrolyzate of geniposide from gardenia fruits. We studied the effect of the amino acid on blue pigment formation and found a linear relationship between the molecular weight of the neutral amino acid and the λ_max of the blue pigment formed. Thin layer chromatographic analysis revealed brilliant skyblue components of the blue pigments formed from glycine, alanine, leucine, phenylalanine and tyrosine. Furthermore, a brilliant skyblue color was obtained by a reverse phase column chromatography (HP-20) of blue pigments formed from glycine, leucine and phenylalanine. The λ_max of these purified pigments lay above 600 nm, and the peaks were sharper than those of crude pigments. After standing for two weeks at 40°C in 40% ethanol solution, the brilliant skyblue pigment formed from genipin and glycine remained stable, losing none of its initial absorbance.     

Monascus secondary metabolites: production and biological activity

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of β-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.     

Synthesis and evaluation of novel 2,3,5-triaryl-4H,2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6-diones for advanced glycation end product formation inhibitory activity

The synthesis of some biologically interesting pyrrolo-isoxazolidine derivatives was accomplished by the 1,3-dipolar cycloaddition reaction of substituted azomethine N-oxides 1 with substituted N-aryl maleimides 2 leading to the formation of new stereoisomeric 2,3,5-triaryl-4H,2,3,3a,5,6,6a-hexahydropyrrolo[3,4-d]isoxazole-4,6-dione derivatives 3 in excellent yields. The synthesized compounds have been screened for their advanced glycation end (AGE) product formation inhibitory activity on the basis of their ability to inhibit the formation of AGEs in the bovine serum albumin (BSA)-glucose assay. All the synthesized compounds have been found to exhibit significant activity against AGE formation.     

Isolation of ommochromes and 3-hydroxykynurenine from the tobacco hornworm,Manduca sexta

A complex of the two red pigments was prepared from epidermis of the fifth instar larvae of the black mutant of the tobacco hornworm according to the method generally available for ommochromes. The primary component, a brick-red pigment (II), was shown to be identical with synthetic l-dihydroxanthommatin by various physico-chemical tests. The other red pigment (I) was similar to pigment (II), except in the i.r. spectra in 1000–1150 cm⁻¹ region and is thought to be also an ommatin. A red pigment identical with pigment (I) was obtained during the chemical oxidation of 3-hydroxy-l-kynurenine. These same two pigments were found in the red epidermis underlying the black cuticle of larvae either allatectomized or neck-ligatured before the critical period for juvenile hormone action during the last larval molt. Also, they were found in the dorsal epidermis of wandering stage larvae. 3-Hydroxykynurenine, the immediate precursor of the ommochrome pigments, was found to accumulate in the epidermis before the synthesis of the ommochromes began.     

Inhibitors of adenosine consuming parasites through polymer-assisted solution phase synthesis of lipophilic 5′-amido-5′-deoxyadenosine derivatives

Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed. Thus, a series of 20 adenosine derivatives with a large lipophilic substituent in N⁶-position were prepared in order to evaluate their potential to inhibit the chloroquine resistant Plasmodium falciparum strain K1 in vitro. The rationale for synthesis of these structures was the high probability of interactions with multiple adenosine associated targets and the assumption that a large hydrophobic N⁶-(4-phenoxy)benzyl substitution should allow the molecules to diffuse across parasite membranes. Starting from readily available inosine, the new compounds were prepared as single isomers using a polymer-assisted acylation protocol enabling the straightforward isolation of the target compounds in pure form. Heterocyclic ring systems were synthesized on-bead on Kenner’s safety-catch linker prior to acylation of the scaffold in solution. Most of the highly pure compounds displayed anti-plasmodial activity in the low micromolar or even submicromolar concentration range.     

Design,synthesis and biological evaluation of six dinuclear platinum(II) complexes

Six dinuclear platinum(II) complexes with a chiral tetradentate ligand, (1R,1′R,2R,2′R)-N¹,N^1′-(1,4-phenylenebis(methylene))dicyclohexane-1,2-diamine, have been designed, synthesized and characterized. In vitro cytotoxicity evaluation of these metal complexes against human A549, HCT-116, MCF-7 and HepG-2 cell lines have been carried out. All compounds showed antitumor activity to HepG-2, HCT-116 and A549. Particularly, compounds A1 and A2 exhibited significant better activity than other four compounds and A2 even showed comparable cytotoxicity to cisplatin against HepG-2 cell line.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Leucine interference in the production of water-soluble red Monascus pigments

The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.Abbreviations mSG Monosodium glutamate - MOPS 3-(N-morpholine)propane sulfonic acid - DCW dry cell weight