Cholesterol as a target for toxins

A mechanism is proposed for the way in which cholesterol facilitates channel formation by polyene antibiotics and bacterial protein toxins. Central elements of the model are: (i) interactions between the ring system of the sterol and rigid elements of the polyene or toxin molecule, and (ii) the specific orientation of cholesterol within the membrane.     

Aromatic dioxygenases: molecular biocatalysis and applications

Aromatic dioxygenases have been found to catalyse single and tandem oxidation reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochemistry and stereochemistry during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilised as precursors for new and alternative routes in chiral synthesis.     

First record of colour polymorphism in the Neotropical apple snail Asolene platae: inheritance mechanism and evidence for multiple paternity

Asolene platae (Ampullariidae) is a dioecious freshwater snail with subaquatic gelatinous egg masses that dwells in the Río de la Plata basin (Argentina). The aim of this study was to describe the inheritance mechanism of the colour variations of the shell and soft parts of this snail, and to study their potential use as a genetic marker. The wild-type phenotype presents dark pigments in the soft parts and in shell bands, whereas the yellow phenotype lacks dark pigments in the soft parts and also most dark bands in the shell, except for a subsutural and a periumbilical band. The data showed that the lack of pigments in A. platae is a recessive homozygotic condition with a simple Mendelian inheritance mechanism. Females of the wild-type phenotype had a higher number of bands than the males. The pigment of the bands of both phenotypes is located in the calcareous matrix of the shell. Using the lack of pigments as a genetic marker we demonstrated the existence of biparental egg masses in A. platae, hitherto known in only one species within the Ampullariidae.     

Structure-based phylogeny of polyene macrolide antibiotic glycosyltransferases

Antibiotic glycosyltransferases (AGts) attach unusual deoxy-sugars to aglycons so antibiotics can exert function. It has been reported that polyene macrolide (PEM) AGts have different evolutionary origin when compared with other polyketide AGts, and our previous analysis have suggested that they could be results of horizontal gene transfer (HGT) from eukaryotes. In this paper, we compared the structures of PEM AGts with structures of eukaryotes and other AGts, and then built models of the representative PEM AGts and GT-1 glycosyltransferases. We also constructed the Neighbor-Joining (NJ) trees based on the normalized Root Mean Square (RMS) distance, the Bayesian tree guided by structural alignments, and carried out analysis on several key conserved residues in PEM AGts. The NJ tree showed a close relationship between PEM AGts and eukaryotic glycosyltransferases, and Bayesian tree further supported their affinity with UDP-glucuronosyltransferases (UGTs). Analysis on key conserved residues showed that PEM AGts may have similar interaction mechanism such as in the formation of hydrogen bonds as eukaryotic glycosyltransferases. Using structure-based phylogenetic approaches, this study further supported that PEM AGts were the result of HGT between prokaryotes and eukaryotes.     

Isolation,taxonomic and fermentation studies on a new strain of Streptomyces arenae var ukrainiana producing a tetraene antibiotic

A Streptomyces strain UK10 was isolated from Ukrainian soil and identified by taxonomical studies as Streptomyces arenae var ukrainiana. HA-2-91 was isolated from the biomass of S. arenae var ukrainiana and is supposedly a polyene macrolide antibiotic belonging to the tetraene group. HA-2-91 showed promising antifungal activity (in vitro) against yeasts and filamentous fungi, including plant pathogens and dermatophytes and was found to be less toxic in mice than nystatin and rimocidin.     

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

Summary The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone² (FZ), toward five cell lines has been determined as measured by early membrane damage (⁵¹Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems. Fungizone^R. Trade mark. E. R. Squibb and Sons. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-02095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Mechanism of anion-cation selectivity of amphotericin B channels

Summary Zero current potential and conductance of ionic channels formed by polyene antibiotic amphotericin B in a lipid bilayer were studied in various electrolyte solutions. Nonpermeant magnesium and sulphate ions were used to independently vary the concentration of monovalent anions and cations as well as to maintain the high ionic strength of the two solutions separated by the membrane. Under certain conditions the channels select very strongly for anions over cations. They are permeable to small inorganic anions. However, in the absence of these anions the channels are practically impermeable to any cation. In the presence of a permeant anion the contribution of monovalent cations to channel conductance grows with an increase in the anion concentration. The ratio of cation-to-anion permeability coefficients is independent of the membrane potential and cation concentration, but it does depend linearly on the sum of concentrations of a permeant anion in the two solutions. These results are accounted for on the assumption that a cation can enter only an anion-occupied channel to form an ionic pair at the center of the channel. The cation is also assumed to slip past the anion and then to leave the channel for the opposite solution. This model with only few parameters can quantitatively describe the concentration dependences of conductance and zero current potential under various conditions.     

An efficient gene transfer system for the pimaricin producer Streptomyces natalensis

Streptomyces natalensis produces the antifungal polyene macrolide pimaricin. Genetic manipulation of its biosynthetic genes has been hampered by the lack of efficient gene transfer systems. We have developed a gene transfer system based on intergeneric conjugation from Escherichia coli. Using this approach, we managed to attain transformation efficiencies of 1 x 10(-4) exconjugants per recipient when using self-replicating vectors such as pHZ1358. The use of integrative vectors such as pSET152 or pSOK804 resulted in significantly lower efficiencies. Site-specific integration or the use of self-replicating plasmids did not affect pimaricin production or the essential functions of S. natalensis. Use of DNA methylation proficient E. coli donor strains resulted in no transformants, indicating the presence of methyl-specific restriction systems in S. natalensis. This methodology will enable easier manipulation of the genes responsible for pimaricin biosynthesis, and could prove valuable for the generation of new designer polyene macrolides with better antifungal activity and pharmacological properties. As an example of the validity of the method, we describe the introduction of Supercos-1-derived cosmid vectors into S. natalensis in order to promote gene replacements by double crossover recombination.