Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.     

The DNA sequence at echinomycin binding sites determines the structural changes induced by drug binding: NMR studies of echinomycin binding to [d(ACGTACGT)]2 and [d(TCGATCGA)]2

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.     

Primary production by benthic microalgae in nearshore marine sediments of Signy Island,Antarctica

Summary During the austral summer of 1987/1988, three 24 h in situ primary productivity measurements were made at a nearshore sublittoral site on the east coast of Signy Island, Antarctica. The first experiment in December, coincided with the peak of the benthic algal bloom as shown by benthic chlorophyll measurements and a primary productivity rate of 700.9 mg carbon m^–2 day^–1. In January, the experiment was undertaken during the peak of the phytoplankton bloom when light intensities reaching the benthos were greatly reduced. A rate of 313.4 mg carbon m^–2 day^–1 was measured, half that of the previous month. In March the phytoplankton bloom had died off, benthic light intensities had increased and production was 391.8 mg m^–2 day^–1. The experiments indicate changes in benthic microalgal activity during the summer, linked to changes in the benthic light climate. Compared with previous measurements of phytoplanktonic activity at Signy, the microphytobenthos seems to be an important source of primary production. A production estimate of 100.9 mg carbon m^–2, for the ice-free summer period, lies within the range of values of results from other polar studies.     

Zebrafish embryology and neural development

The zebrafish is rapidly increasing in popularity with developmental biologists. Driving this interest are the elegant methods for in vivo observations and recovery of early developmental mutations. The past year has seen the introduction of additional methods for in vivo manipulation of identified cells and the application of these methods to mutant analysis.     

Synergism within polyhexamethylene biguanide biocide formulations

Polyhexamethylene biguanides (PHMB) are mixtures of polymeric biguanides with an average polymer length (n) of 5, but containing high (n greater than 15, mol. wt 3300) and low molecular weight material (n = 2, mol. wt 400). Studies involving discrete molecular weight fractions of PHMB have shown that antimicrobial activity of PHMB increases with increasing polymer length. Cell suspensions which had not been subjected to centrifugation and/or washing during their preparation were employed. Whilst activity was still observed to increase with n, the trend was much reduced as n exceeded six. Centrifugation and washing of cells markedly increased the activity of high but not low molecular weight materials and corresponded to losses upon centrifugation of envelope lipopolysaccharide (LPS). Such envelope LPS represented high affinity binding sites on the surfaces of the cells. Combinations of various molecular weight fractions of PHMB were evaluated against filter-washed cells and revealed a profound synergy between extremes of polymer length.     

Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Injury and recovery of Bacillus megaterium from mild chlorhexidine treatment

Treatment of Bacillus megaterium cell suspensions with 12 /μmol/1 chlorhexidine diacetate for 5 min led to an approximate 50%, reduction in viability when plated onto tryptone soya agar (TSA). Fifty percent of the surviving fraction were unable to form colonies on TSA containing 5.5% w/v KCI. Such loss of KCl tolerance is indicative of membrane damage, and was recovered within 30 min of incubation in tryptone soya broth (TSB). Multiplication of the damaged organisms did not recommence in this medium until after 60 min. Inclusion of inhibitors of respiration, and of protein, RNA and DNA synthesis in the TSB recovery medium did not significantly affect either the rate or extent of the recovery of KCl tolerance by damaged organisms.