Mechanism of the antibiotic action pyocyanine.

Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.     

Identification of a rosette-enriched chromatin fraction from mouse fibroblast nuclei

We have characterized two components of DNA isolated from mouse L-M cell nuclei. These components, designated as HMW (high molecular weight) and VHMW (very high molecular weight) DNA, were characterized by rate zonal sedimentation, agarose gel electrophoresis, and for protein content. Our electron micrographs revealed that HMW-DNA contained mainly linear molecules with few single rosette structures, while the VHMW-DNA was enriched in rosettes, many of which were significantly larger and linked together in multimeric structural forms. The VHMW-DNA component was also enriched for residual protein, which we believe represents the core of the rosette. The characteristics of this residual protein are consistent with reported findings of the most tightly bound proteins. The rosette conformation does not appear to be an artifact of microscopy or of an aggregate nature for several reasons: (i) rosettes are preferentially found in the VHMW-DNA component; (ii) further manipulation or purification of the DNA disrupts the rosette structure and produces linear fragments; (iii) the amount of proteinaceous material at the core of the rosette is diminished when the DNA is further purified; and (iv) treatment of intact nuclei with a novel bisamine reagent putatively crosslinks DNA in vivo and minimizes the disruption of rosettes by shear. We believe this separation of chromatin is critical to establish the architectural forms of euchromatin and heterochromatin of interphase DNA in the eucaryotic system. Once established, fractionated chromatin can be used to identify specifically expressed or repressed genes with linear form DNA and rosette form DNA. We discuss rosettes as derivatives of chromosomal domains that retain structural features because of residual peptide elements.     

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

Possibility of detecting pyocyanine in Pseudomonas aeruginosa cells

Pyocyanin can be detected in the cells of Pseudomonas aeruginosa using UV and IR spectroscopy of disturbed complete inner reflection (DCIR). Intact cells of the parent strain liberating the pigment into the cultural broth and mutant cells lacking the ability contain pyocyanin within the cells. Occasionally, pyocyanin can be detected in the outer layers of the cells, which is more typical of the parent strain. In the freshly isolated fractions of the parent strain cellular walls, pyocyanin seems to be pesent in the bound state that has changed significantly its structural organization. In due course, the hypothetical complex pyocyanin--cellular wall decomposes to yield an "oxidized" pigment similar to that liberated into the cultural broth. the cell wall of the mutant possesses the properties of "oxidized" pyocyanin immediately after isolation of the fraction. The pigment cannot be identified in the fractions of cytoplasmic membranes; pyocyanin is present in the "oxidized" state in the fractions of cytoplasm for the cells of both types. The paper discusses the role of the permeability of cytoplasmic membranes in the transport of pyocyanin from the cytoplasm into the cellular wall of the bacterium and then into the surrounding medium.     

The physiologic role of pyocyanine synthesized by Pseudomonas aeruginosa

The physiological role of pyocyanine for Pseudomonas aeruginosa was studied. Its synthesis was shown to commence at the retardation growth phase. Pyocyanine was accumulated only in the growth medium. The addition of 2,6-dichlorophenolindophenol accepting the reducing equivalents from coenzyme Q and transferring them to cytochrome c inhibited the pigment accumulation. This was indicative of the connection between pyocyanine synthesis and the level of the reducing equivalents in the cells. Pyocyanine did not accept the reducing equivalents from coenzyme Q in the respiratory chain of P. aeruginosa. Only reduced pyridine nucleotides served as substrates for pyocyanine in the reaction of autooxidation. The kinetic parameters of this reaction and the affinity of NADH dehydrogenase for the substrate were measured. The kinetic data were analysed to show that, under the physiological conditions, pyocyanine could not apparently compete with the respiratory chain for the reducing equivalents and hence directly regulate the level of NAD(P)H in P. aeruginosa cells. In order to keep the oxidising activity at a level necessary for the cells, the latter decreased the content of the reducing equivalents either by synthesizing pyocyanine or owing to the activity of cyanide-resistant oxidase. These processes of releasing the reducing equivalents are in a reciprocal relationship.     

Phenotype recognition of pyocyanine mutants in pseudomonas aeruginosa

Certain classes of pyocyanine mutants in Pseudomonas aeruginosa escape detection when screened in the presence of wild-type or other mutant cells. A technique is described for recognizing mutant phenotypes after cells are in individual agar wells. The procedure eliminates cross-feeding phenomena as well as the masking of mutant phenotypes by diffusing pyocyanine produced by nearby clones.     

Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

Enhancement of synaptic vesicle attachment to the plasma membrane fraction by copper

Synaptic vesicles from rat brain were labeled with¹²⁵I, and the association of the vesicles with other subcellular components of brain was examined using a centrifugation assay. Copper at micromolar concentrations enhances the binding of the vesicles to the synaptic membrane as well as other fractions. Magnesium, Ca²⁺, and calmodulin with Ca²⁺ are ineffective. There is virtually no binding of synaptic vesicles to the microtuble fraction and only a slight enhancement with Cu²⁺. These findings support the hypothesis that Cu may serve as a bridge between synaptic vesicles and the plasma membrane.     

Identification of hypoxanthine as the major compoenet of a chromatographic fraction of transfer factor.

A chromatographic fraction of transfer factor capable of transferring delayed hypersensitivity has been shown by us and recently confirmed by others to elute from a Sephadex G-25 column in a peak after the total column volume. The chemical composition of this fraction (TFc) was determined using fractions prepared in the same manner as preparations previously shown to have biologic activity. The major component in this fraction has been identified as hypoxanthine by identical behavior in paper, ion exchange, and gel permeation chromatography systems as well as by comparison of ultraviolet (uv), nuclear magnetic resonance (NMR) and infrared (IR) spectra of the column purified material with the spectra of crystalline hypoxanthine. In addition, the uv absorption of TFc can be converted quantitatively to uric acid by incubation with xanthine oxidase. Possible explanations for this observation include: 1) the material responsible for transfer of delayed hypersensitivity may be degraded and absent from these preparations, 2) the antigen specific component may constitute a very minor component of this fraction while hypoxanthine is responsible for some of the reported non-specific effects of transfer factor, and 3) the antigen specific transfer factor material does not elute in this fraction.