Immuno-modulating properties of a macrolide antibiotic "Z-factor"]

Zi-Factor is a trade mark of azithromycin made in Russia by ZAO Veropharm. The in vitro modulating action of Zi-Factor (ZF) on neutrophil functional activity and production of immunity mediators was studied. The direct modulating effect of ZF in a concentration of 50 mcg/ml corresponding to the single therapeutic dose of 500 mg/70 kg body weight on the neutrophil oxidase activity evident from increased production of active oxygen and higher myeloperoxidase activity was shown. The immunomodulating effect of ZF on modulation of intracellular oxidative metabolism depended on the initial state of the phagocyte system (secondary insufficiency or activation). When the neutrophil oxidase system is exhausted and there is no response to the antigen stimulus, it is advisable to combine the use of ZF with immunomodulating therapy.     

Enhancement of endogenous corticosterone levels by a macrolide antibiotic, roxithromycin in mice

The influence of roxithromycin (RXM), a macrolide antibiotic, on endogenous corticosterone (CS) levels was examined in BALB/c mice. Mice were sensitized intraperitoneally with two doses of Keyhole Limpet Hemocyanin at 1 week intervals. Mice were given orally 2.5 mg/kg RXM once a day for 14 days starting 7 days after the first sensitization. RXM administration caused markedly increase in endogenous plasma CS levels which was peaked at 60 min after the administration. However, josamycin did not influence on endogenous CS levels in plasma. Injection of dexamethasone inhibits the plasma CS hyperproduction induced by RXM treatment.     

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm×75 μm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.     

Inhibition of oleandomycin synthesis by glucoses added during fermentation

Oleandomycin biosynthesis by Streptomyces antibioticus is repressed by glucose added to the growth medium in the process of fermentation. Phosphotransferase involved in the synthesis of acetyl CoA and propionyl CoA (the precursors of the antibiotic macrolactone ring) is neither inhibited nor repressed, and the substrate specificity of the enzyme does not change. The content of cAMP in the mycelium of S. antibioticus does not change significantly when either glucose or sucrose is added to the medium 24 h after the inoculation whereas the content of exogenous cAMP rises abruptly 24 h after glucose addition. At the same time, the medium becomes much more acidic and the content of protein in the mycelium rises noticeably. Consequently, cAMP may be involved in the regulation of the culture growth.     

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k_c, from 23.1 ± 3.5 to 10.6 ± 4.5 k_BT) and the apparent area compressibility modulus (K_app, from 176 ± 35 to 113 ± 25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.     

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.     

Enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase

Enzymatic glycosidation using sugar oxazolines 1-3 having a carboxylate group as glycosyl donors and compounds 4-6 as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor. The N,N'-diacetyl-6'-O-carboxymethylchitobiose oxazoline derivative 1 was effectively glycosidated, under catalysis by the enzyme, with methyl N,N'-diacetyl-beta-chitobioside (4), pent-4-enyl N-acetyl-beta-D-glucosaminide (5), and methyl N-acetyl-beta-D-glucosaminide (6), affording in good yields the corresponding oligosaccharide derivatives having 6-O-carboxymethyl group at the nonreducing GlcNAc residue. The N,N'-diacetyl-6-O-carboxymethylchitobiose oxazoline derivative 2 was subjected to catalysis by the enzyme catalysis; however, no glycosidated products were produced through the reactions with 4, 5, and 6. Glycosidation reactions of the beta-d-glucosyluronic-(1-->4)-N-acetyl-D-glucosamine oxazoline derivative 3 proceeded with each of the glycosyl acceptors, giving rise to the corresponding oligosaccharide derivative having a GlcA residue at their nonreducing termini in good yields.     

Drug resistance in Staphylococcus aureus. Induction of macrolide resistance by erythromycin, oleandomycin and their derivatives.

Antibacterial and inducer activities concerning inducible macrolide resistance in Staphylococcus aureus were investigated using 32 erythromycin, oleandomycin and other macrolide antibiotic derivatives and analogues. The macrolides were classified into five groups from very high to none according to their inducer activity.     

Adjuvant effect of a 14-member macrolide antibiotic on DNA vaccine

Macrolide antibiotics have unique immunomodulatory actions apart from their antimicrobial properties. We examined the effect of erythromycin (EM), a 14-member macrolide, on the immune response to a DNA vaccine that induces a T-helper-1 (Th1)-biased immune response through a Th1-promoting adjuvant effect of unmethylated CpG motifs within plasmid DNA. EM enhanced Th1 responses in plasmid DNA-immunized mice as measured by antigen-specific IgG2a antibody production, interferon-gamma production by antigen-specific CD4(+) T cells, and cytotoxic T lymphocyte responses. EM augmented the accessory cell activity of unmethylated CpG DNA-stimulated antigen-presenting cells (APCs), suggesting that EM enhances Th1 responses to a DNA vaccine, possibly through augmentation of accessory cell activity of APCs stimulated with CpG motifs within plasmid DNA.     

Structure and anomeric configuration of the C-nucleoside analogs obtained by dehydration of 7-deoxy-l-manno-2-heptulose phenylosazone

Dehydration of 7-deoxy-l-manno-2-heptulose phenylosazone with methanolic sulfuric acid afforded two 3,6-anhydro-osazone derivatives (2 and 3). Refluxing the anhydro-osazones with copper sulfate gave two C-nucleoside analogs, namely, 4-(5-deoxy-α-l-arabinofuranosyl)-2-phenyl-ν-triazole (4) and 4-(5-deoxy-β-l-arabinofuranosyl)-2-phenyl-ν-triazole (5). The structure and anomeric configurations of 2, 4, and 5 were determined by n.m.r. spectroscopy. The preponderant conformation of 4 and 5, and the mass spectra of 2, 4, and 5 are discussed.     

Preparation of anomeric pairs of 1-thioglycosides: use of anomerization catalyzed by boron trifluoride

Anomeric pairs of some alkyl 1-thioaldopyranosides of d-galactose, d-glucose, d-mannose, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, and l-fucose were prepared. The per-O-acetylated, 1,2-trans anomers of 6-(trifluoroacetamido)hexyl 1-thioaldopyranosides and 5-(methoxycarbonyl)pentyl 1-thioaldopyranosides were anomerized with boron trifluoride in dichloromethane. The anomeric mixtures were then separated by chromatography, using columns of either silica gel or an ion-exchange resin. De-blocking of the separated compounds provided pure anomers of 6-aminobexyl 1-thioaldopyranosides or 5-carboxypentyl 1-thioaldopyranosides. The aglycons of the latter glycosides were further extended by reaction with aminoacetaldehyde diethyl acetal, which, after deacetalization of the products, provided an ω-aldehydo group. These series of glycosides could be readily coupled to proteins or solid matrices.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Production of the macrolide antibiotic tylosin in cyclic fed-batch culture

Tylosin-producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate-glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (q(tylosin), mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high q(tylosin) had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed-batch fermentations with glutamate and glucose being fed to the culture in cyclic square-wave profiles with methyloleate in excess showed several-fold increase in final q(tylosin) and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed-batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when alpha-ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of q(tylosin) up to 240 h.