Effects of coumarin, thiopurines, and pyronin Y on amplification of phleomycin-induced death and deoxyribonucleic acid breakdown in Escherichia coli

Phleomycin (/=10 mug of phleomycin per ml were observed among 10(11)E. coli B cells screened, such mutants occurred with a frequency of 10(-6) to 10(-7) among cultures resistant to 1 to 2 mug of phleomycin per ml. These double mutants were cross-resistant to phleomycin plus caffeine. The amplifying compounds, though structurally dissimilar, shared the common characteristic of binding selectively to denatured DNA as measured by equilibrium dialysis methods. The implications of these observations in supporting a model of phleomycin amplification proposed previously (6) and their utility in providing a logic for developing a new class of antibiotics are discussed.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1

The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k(cat)/K(m) = 12800 ± 600 M(-1) s(-1)) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k(cat)/K(m) = 2000 ± 100 M(-1) s(-1)) and also oxidized Mn(II) (k(cat)/K(m) = 25.1 ± 0.1 M(-1) s(-1)). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = (5)/(2)) Fe(III) in the resting state, DypA had a rhombic high-spin signal (g(y) = 6.32, g(x) = 5.45, and g(z) = 1.97) while DypB had a predominantly axial signal (g(y) = 6.09, g(x) = 5.45, and g(z) = 1.99). Moreover, DypA reacted with H(2)O(2) to generate an intermediate with features of compound II (Fe(IV)═O). By contrast, DypB reacted with H(2)O(2) with a second-order rate constant of (1.79 ± 0.06) × 10(5) M(-1) s(-1) to generate a relatively stable green-colored intermediate (t(1/2) ～ 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [Fe(IV)═O Por(?)](+) species. The X-ray crystal structure of DypB, determined to 1.4 ? resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H(2)O(2). A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind Mn(II). Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.     

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.     

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (k_cat/K_M ∼10⁶ M⁻¹ s⁻¹), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates’ binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate’s alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.     

Lack of a Functioning P2X7 Receptor Leads to Increased Susceptibility to Toxoplasmic Ileitis

Background

Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis.

Principal Findings

Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice.

Conclusions

Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.     

Transmission of Toxoplasma: clues from the study of sea otters as sentinels of Toxoplasma gondii flow into the marine environment

Toxoplasma gondii affects a wide variety of hosts including threatened southern sea otters (Enhydra lutris nereis) which serve as sentinels for the detection of the parasite's transmission into marine ecosystems. Toxoplasmosis is a major cause of mortality and contributor to the slow rate of population recovery for southern sea otters in California. An updated seroprevalence analysis showed that 52% of 305 freshly dead, beachcast sea otters and 38% of 257 live sea otters sampled along the California coast from 1998 to 2004 were infected with T. gondii. Areas with high T. gondii exposure were predominantly sandy bays near urban centres with freshwater runoff. Genotypic characterisation of 15 new T. gondii isolates obtained from otters in 2004 identified only X alleles at B1 and SAG1. A total of 38/50 or 72% of all otter isolates so far examined have been infected with a Type X strain. Type X isolates were also obtained from a Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Molecular analysis using the C8 RAPD marker showed that the X isolates were more genetically heterogeneous than archetypal Type I, II and III genotypes of T. gondii. The origin and transmission of the Type X T. gondii genotype are not yet clear. Sea otters do not prey on known intermediate hosts for T. gondii and vertical transmission appears to play a minor role in maintaining infection in the populations. Therefore, the most likely source of infection is by infectious, environmentally resistant oocysts that are shed in the feces of felids and transported via freshwater runoff into the marine ecosystem. As nearshore predators, otters serve as sentinels of protozoal pathogen flow into the marine environment since they share the same environment and consume some of the same foods as humans. Investigation into the processes promoting T. gondii infections in sea otters will provide a better understanding of terrestrial parasite flow and the emergence of disease at the interface between wildlife, domestic animals and humans.     

An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain.     

Low temperature, gibberellin and acid lipase activity in removal of apple seed dormancy

The activity of acid lipase and the level of gibberellin A₄ (GA₄) were determined in apple embryos excised from seeds after different time periods of stratification and subsequently cultured in darkness at 4°C or at 25°C. Enzyme activity and GA₄ content were higher at 4°C. Exogenous gibberellin stimulated lipase activity, while AMO-1618, an inhibitor of gibberellin biosynthesis, inhibited, to the same degree, both the enzyme activity and the GA₄ accumulation. The involvement of GA₄ and lipolytic enzymes in cold-mediated removal of embryonal dormancy has been discussed and compared with the role of these two factors in light-stimulated germination of dormant apple embryos, described earlier (Smoleńska and Lewak 1974).     

Gill function in an elasmobranch

Summary Highly efficient oxygen uptake in elasmobranchs, as indicated by frequent excess of over has previously been ascribed to the operation of multicapillary rather than counter-current gas exchange by the gills. Analysis of models shows that, at maximum efficiency, a multicapillary system cannot account for values of greater than . In Port Jackson sharks Heterodontus portusjacksoni) commonly exceeds , which indicates the operation of a functional counter-current at the respiratory surface. The anatomical basis of this counter-current is provided by the demonstration that a continuous flow of water passes between the secondary lamellae into septal canals and thence via the parabranchial cavities to the exterior.Queen Elizabeth II Fellow.