Streptomyces hygroscopicus UFPEDA 3370: A valuable source of the potent cytotoxic agent nigericin and its evaluation against human colorectal cancer cells.

Streptomyces hygroscopicus UFPEDA 3370 was fermented in submerged cultivation and the biomass extract was partitioned, obtaining a fraction purified named EB1. After purification of EB1 fraction, nigericin free acid was obtained and identified. Nigericin presented cytotoxic activity against several cancer cell lines, being most active against HL-60 (human leukemia) and HCT-116 (human colon carcinoma) cell lines, presenting IC50 and (IS) values: 0.0014 µM, (30.0) and 0.0138 µM (3.0), respectively. On HCT-116, nigericin caused apoptosis and autophagy. In this study, nigericin was also screened both in vitro and in silico against a panel of cancer-related kinases. Nigericin was able to inhibit both JAK3 and GSK-3ß kinases in vitro and its binding affinities were mapped through the intermolecular interactions with each target in silico.

The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC50 of 2 µM, and TbAQP3 to a much lesser extent (IC50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

Evolutionary stability of antibiotic protection in a defensive symbiosis.

The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Static magnetic field (SMF) sensing of the P(723)/P(689) photosynthetic complex.

Moderate intensity SMF have been shown to act as a controller of the protic potential in the coherent milieu of the thylakoid membranes. SMF of the order of 60-500 mT induces memory-like effect in photosystem I (PSI, P723) emission with a correlated oscillation of photosystem II (PSII, P689) fluorescence emission at a temperature of 77 K. The observed magnetic perturbation that affects the thylakoid photon capture circuitry was also found to be associated with the bio-energetic machinery of the thylakoid membranes. At normal pH, SMF causes an enhancement of PSI fluorescence emission intensity (P723/P689 > 1), followed by a slow relaxation on the removal of SMF. The enhancement of the PSI fluorescence intensity also occurs under no-field condition, if either the pH of the medium is lowered, or protonophores, such as carbonyl cyanide chlorophenylhydrazine or nigericin are added (P723/P689≥ 2). If SMF was applied under such a low pH condition or in the presence of protonophore, a reverse effect, particularly, a reduction of the enhanced PSI emission was observed. Because SMF is essentially equivalent to a spin perturbation, the observed effects can be explained in terms of spin re-organization, illustrating a memory effect via membrane re-alignment and assembly. The mimicry of conventional uncouplers by SMF is also notable; the essential difference being the reversibility and manoeuvrability of the latter (SMF). Finally, the effect implies numerous possibilities of externally regulating the photon capture and proton circulation in the thylakoid membranes using controlled SMF.

Investigation of the maximum quantum yield of PS II in Haematococcus pluvialis cell cultures during growth: effects of chemical or high-intensity light treatment.

In this study, we investigated the increase in photosynthetic quantum yield that occurs in advance of increased microalgal growth. Haematococcus pluvialis was cultivated under normal conditions; the number of cells, the maximum quantum yield of photosystem II (F(v)/F(m)), and optical density were measured. We observed an increase in F(v)/F(m) approximately 72h prior to the cell growth phase. To confirm the relationship between photosynthetic yield and growth, samples were treated with several chemicals under high-intensity light illumination and control conditions to inhibit photosystem II and induce a decrease in the quantum photosynthetic yield. The samples were exposed to high-intensity light at an irradiance of 400µmol photonsm(-2)s(-1) for varied amount of time and were treated with chemicals such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea, nigericin sodium salt and valinomycin. We observed that both the photooxidation of photosystem II reaction centers and the formation of transmembrane electrochemical gradients led to an initial decrease in fluorescence yield after the onset of high-intensity light illumination. We also observed that treatment of high-intensity light illuminated cells with antibiotics after adaptation to moderate light intensities caused a difference in photosynthetic activity. In conclusion, the maximum quantum yield of photosystem II is obtained prior to the cell growth phase and can therefore be used as a prediction parameter for cell growth.

Nigericin-mediated liposome loading of topotecan: is nigericin a potential drug release regulator?

Topotecan was loaded into large unilamellar vesicles using nigericin-generated pH gradient or with triethylammonium (TA) ion gradient. Despite that in both loading methods, the encapsulated counter ion was 5-sulfosalicylate (5ssa), the resultant formulations exhibited distinct properties. In NaCl-containing release buffer, vesicles prepared with nigericin/Na(+) system could release topotecan at a faster rate than 5ssa-TA vesicles, whereas in the absence of Na(+), there was no difference in the drug release kinetics. In plasma, both formulations could prolong the circulation halftime of topotecan, but 5ssa-TA vesicles were more able to stabilize the encapsulated topotecan, as evidenced by the increased t(1/2) and decreased conversion of lactone to carboxylate form. However, the improved drug retention did not mean the elevated safety and efficacy. In L1210 ascitic tumor model, the administration of 5ssa-TA vesicles at 10mg/kg induced the early death of ∼60% mice at 6-7 days. In contrast, the treatment with nigericin/Na(+) vesicles at the same dose level resulted in a mean survival time of ∼18.0 days, ∼1.38-fold of that of free topotecan. In addition, the formulation was safer than free topotecan. The results indicated that nigericin could be used as release regulator to optimize drug release kinetics, resulting in safe and efficacious liposome-based topotecan formulation. The results were promising since no liposome topotecan formulations with reduced toxicity were reported.

Copper ion-mediated liposomal encapsulation of mitoxantrone: the role of anions in drug loading, retention and release.

Besides pH gradient, other transmembrane gradients such as metal ion gradient could be also employed to load drugs into liposomes. In pH gradient method, anions have an important role since they could form specific aggregates with drugs, and then affect drug release kinetics from vesicles. To explore the role of anions in metal ion gradient method, copper ion-mediated mitoxantrone (MIT) loading was investigated systematically. When empty liposomes exhibiting a transmembrane copper ion gradient (300 mM) were mixed with MIT in a molar ratio of 0.2:1, after 5 min incubation at 60 degrees C, >95% MIT could be loaded into vesicles and the encapsulation was stable, regardless of the kinds of anions and initial intraliposomal pH values. The encapsulation ratio decreased with increased MIT/lipid molar ratio. But even when the molar ratio increased to 0.4, >90% encapsulation could still be achieved. In the presence of nigericin and ammonium, the drug loading profiles were affected to different degree with respect to both drug loading rate and encapsulation ratio. Relative to CuSO(4)-containing systems, CuCl(2) mediated MIT loading was unstable. Both nigericin and ammonium could alter the absorption spectra of liposomal MITs loaded with CuSO(4) gradient. In vitro release studies were performed in glucose/histidine buffer and in 50% human plasma using a dialysis method. In both of release media, CuCl(2)-containing vesicles displayed rapid release kinetics in comparison with CuSO(4) systems; and during the experiment period, MIT was lost from the vesicles continuously. When the formulations were injected into BDF1 mice at a dose of 4 mg/kg, all the liposomal formulations exhibited enhanced blood circulation time, with half-life values of 6.8-7.2h, significantly compared to the rapid clearance of free-MIT. In L1210 ascitic model, CuCl(2) formulation was more therapeutically active than CuSO(4) formulation. At a dose of 6 mg/kg, the treatment with CuCl(2) formulation resulted in a median survival time of 21 days, considerably larger than that of CuSO(4) groups (15 days). Based on these data, it was concluded that during the drug loading process, a dynamic transmembrane pH gradient is generated and intraliposomal pH might affect the complexation manner in which Cu(2+) binds MIT. Owing to the presence of pH gradient, after the accumulation within vesicles, a part of MIT will be protonated and precipitated by sulfate. Accordingly, the aggregation status of MIT inside CuSO(4) system was more complicated than that in CuCl(2) vesicles. The difference in physical status of MIT aggregates affects not only the drug release rate, but also their therapeutic effects.

Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137.

Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comparison of the nigericin biosynthetic genes with their counterparts in the biosynthetic clusters for other polyketides has prompted a significant revision of the proposed common pathway for polyether biosynthesis. In particular, we present evidence that in nigericin, nanchangmycin, and monensin, an unusual ketosynthase-like protein, KSX, transfers the initially formed linear polyketide chain to a discrete acyl carrier protein, ACPX, for oxidative cyclization. Consistent with this, deletion of either monACPX or monKSX from the monensin gene cluster effectively abolished monensin A biosynthesis.

Soraprazan: setting new standards in inhibition of gastric acid secretion.

After treatment of millions of patients suffering from gastroesophageal reflux disease (GERD) and other acid-related ailments with proton pump inhibitors, there are still unmet medical needs such as rapid and reliable pain relief, especially for nocturnal acid breakthrough. In this work, we introduce and characterize the biochemistry and pharmacology of the potassium-competitive acid blocker (P-CAB) soraprazan, a novel, reversible, and fast-acting inhibitor of gastric H,K-ATPase. Inhibitory and binding properties of soraprazan were analyzed together with its mode of action, its selectivity, and its in vivo potency. This P-CAB has an IC(50) of 0.1 microM if measured with ion leaky vesicles and of 0.19 microM in isolated gastric glands. With a K(i) of 6.4 nM, a K(d) of 26.4 nM, and a B(max) of 2.89 nmol/mg, this compound is a highly potent and reversible inhibitor of the H,K-ATPase. Soraprazan shows immediate inhibition of acid secretion in various in vitro models and in vivo and was found to be more than 2000-fold selective for H,K-ATPase over Na,K- and Ca-ATPases. Soraprazan is superior to esomeprazole in terms of onset of action and the extent and duration of pH elevation in vivo in the dog. Rapid and consistent inhibition of acid secretion by soraprazan renders the P-CABs a promising group of compounds for therapy of GERD.

Copper-topotecan complexation mediates drug accumulation into liposomes.

These studies describe the role of transition metal ions in the liposomal encapsulation of topotecan. Liposomes (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CH) (55:45, mole ratio)) were prepared with manganese (Mn), copper (Cu), zinc (Zn) or cobalt (Co) ion gradients (metal inside). Subsequently, topotecan was added to the liposome exterior (final drug-to-lipid ratio (mol/mol) of 0.2) and drug encapsulation was measured as a function of time and temperature. No drug loading was achieved with liposomes containing Co or Zn. Topotecan could be encapsulated into Mn-containing liposomes only in the presence of the ionophore, A23187 suggesting that a transmembrane pH gradient was necessary. However, Cu-containing liposomes, in the presence or absence of an imposed pH gradient, efficiently encapsulated topotecan. It has been reported that Cu(II) can form transition metal complexes with camptothecin; therefore, the Cu-topotecan interaction was characterized in solution as a function of pH. These investigations demonstrated that topotecan inhibited formation of an insoluble Cu hydroxide precipitate. Cryo-TEM analysis of the topotecan-loaded Cu liposomes showed electron-dense intravesicular precipitates. Further studies demonstrated that only the active lactone form of the drug was encapsulated and this form predominated in Cu-containing liposomes. Copper complexation reactions define a viable methodology to prepare liposomal camptothecin formulations.

A combined experimental and quantum chemical study on the putative protonophoric activity of thiocyanate.

Inhibition of gastric acid secretion by thiocyanate is explained by a protonophoric mechanism assuming that thiocyanate induces a H(+) back flux from the acidic gastric lumen into the parietal cells of gastric mucosa. Protonophoric activity of thiocyanate was examined by swelling measurements using rat liver mitochondria and theoretically by quantum chemical methods. Mitochondria suspended in K-thiocyanate medium plus nigericin (an H/K-exchanger) swelled when the medium pH was acidic, indicating that SCN(-) initiates a transfer of H(+) across the inner membrane. To rationalize the protonophoric activity of thiocyanate, we considered the dehydration of SCN(-) to be critical for transmembranal H(+) transfer. For modeling this process, various hydrate clusters of SCN(-) and Cl(-) were generated and optimized by density functional theory (DFT) at the B3-LYP/6-311++G(d,p) level. The cluster hydration energy was lower for SCN(-) than for Cl(-). The total Gibbs free energies of hydration of the ions were estimated by a hybrid supermolecule-continuum approach based on DFT. The calculated hydration energies also led to the conclusion that SCN(-) is less efficiently solvated than Cl(-). Due to the easier removal of the hydration shell of SCN(-) relative to Cl(-), SCN(-) is favored in going across the membrane, giving rise to the protonophoric activity.

The ionophore nigericin transports Pb2+ with high activity and selectivity: a comparison to monensin and ionomycin.

The K(+) ionophore nigericin is shown to be highly effective as an ionophore for Pb(2+) but not other divalent cations, including Cu(2+), Zn(2+), Cd(2+), Mn(2+), Co(2+), Ca(2+), Ni(2+), and Sr(2+). Among this group a minor activity for Cu(2+) transport is seen, while for the others activity is near or below the limit of detection. The selectivity of nigericin for Pb(2+) exceeds that of ionomycin or monensin and arises, at least in part, from a high stability of nigericin-Pb(2+) complexes. Plots of log rate vs log Pb(2+) or log ionophore concentration, together with the pH dependency, indicate that nigericin transports Pb(2+) via the species NigPbOH and by a mechanism that is predominately electroneutral. As with monensin and ionomycin, a minor fraction of activity may be electrogenic, based upon a stimulation of rate that is produced by agents which prevent the formation of transmembrane electrical potentials. Nigericin-catalyzed Pb(2+) transport is not inhibited by physiological concentrations of Ca(2+) or Mg(2+) and is only modestly affected by K(+) and Na(+) concentrations in the range of 0-100 mM. These characteristics, together with higher selectivity and efficiency, suggest that nigericin may be more useful than monensin in the treatment of Pb intoxication.

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles.

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.

[Isolation and partial characterization of NAD+ kinase from Streptomyces albogriseolus 444]. / Vydelenie i chastichnaya kharakteristika NAD+ kinazy Streptomyces albogriseolus 444

NAD+ kinase was isolated from the culture of Streptomyces albogriseolus 444. an organism producing an antibiotic complex. The enzyme was partially characterized. Its molecular weight is 141219 Da.

Application of fast atom bombardment combined with tandem mass spectrometry to the structural elucidation of O-demethylabierixin and related polyether antibiotics.

O-Demethylabierixin, a new polyether antibiotic, was isolated from a Streptomyces spp. Its structure elucidation was carried out with fast atom bombardment mass spectrometry (FAB-MS) and fast atom bombardment mass spectrometry-mass spectrometry (FAB-MS/MS) by comparing spectral patterns with those of structurally similar polyether compounds, nigericin and abierixin. The collision-induced dissociation (CID) of sodium-adduct and deprotonated molecular ions produced many cyclic ether rings-opened product ions via a series of the dissociative processes. Because of the negative charge of the carboxylate group at the end of the molecule, the charge-remote fragmentation pattern in the negative CID spectra of deprotonated molecular ions was very helpful for complete identification of product ions which are characteristic for cyclic ether structures. From this study, the new polyether antibiotic was identified to be O-demethylabierixin in which the methoxy group of six-membered ether ring in abierixin was replaced by the hydroxy group.

Human buccal epithelial cells as a potential biochemical predictor of essential hypertension: identification of key cellular processes.

1. Human cheek cell Na+/H+ antiporter activity (measured as the rate of proton-dependent 22Na+ uptake) was determined in seven normotensive (NT) and four hypertensive (HT) subjects following preincubation of cheek cells with a low molecular weight fraction isolated from NT saliva together with the ionophore, nigericin. 2. Cheek cells preincubated in this manner exhibited greater Na+/H+ antiporter activity with the mean values being 4.2 nmol Na+.mg protein.5 min for the NT group and 1.7 for the HT group. 3. It is possible that stimulation of Na+ transport is due to cellular accumulation of K+ ions during preincubation which, in the presence of the K+/H+ selective ionophore, nigericin, can cause cellular reacidification promoting further 22Na+ uptake via the Na+/H+ antiporter.

23Na- and 1H-NMR studies of the action of chlorpromazine and imipramine on nigericin-mediated Na+ transport across phosphatidylcholine vesicular membranes.

In order to elucidate the action of chlorpromazine (CPZ) and imipramine (IMP) on nigericin-mediated Na+ transport across phosphatidylcholine vesicular membranes, 23Na nuclear magnetic resonance was applied to the exchange system of Na+ ions present at the same concentration inside and outside unilamellar vesicles. CPZ and IMP added to the vesicles in micromolar concentrations produced an equal increase in the carrier-transport rate. The kinetic analysis, together with 1H-NMR observations of the reduction in membrane fluidity produced by the drugs and on the direct interaction between drugs and nigericin, allowed us to conclude that the drug-induced promotion of transport occurred not from the formation step of the Na(+)-nigericin complex nor from its diffusion step, but from its dissociation step. The formation of an adduct between drug and nigericin could be the cause of the drug effect and this proceeded much more efficiently at a membrane-water interface (stability constant Kb; 3 x 10(5) M-1) than in methanol (Kb; 5 x 10(2) M-1). The reason for the difference is also discussed.

Secondary metabolites by chemical screening. 17. Nigericinol derivatives: synthesis, biological activities, and modeling studies.

The synthesis and the biological activity of C-1-reduced nigericin derivatives (nigericinols) are described and discussed. The dichloronigericinol 7 impressively demonstrated that the C-1 carboxylic acid moiety was not required for a distinct activity against bacteria and viruses. Based on the correlation between K+/H+ antiport activities and antibacterial activities it was deduced that the mode of action of the described nigericinols are related to their ionophoric properties. Molecular modeling studies showed that the efficiency of the nigericinols as ionophores correlates, qualitatively, with the probability of forming a cyclic structure, with the exception of 7.