Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species

Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NO*) by activated macrophages. Subsequent reaction of NO* with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2-), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2'-deoxy-8-oxoguanosine. Alternatively, the reaction of NO and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from dG; 2'-deoxyinosine (dI) from dA; and 2'-deoxyuridine (dU) from dC, in addition to abasic sites and dG-dG cross-links. The presence of both ONOOCO2- and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (k(cat)/Km 29 x 10(-6), 21 x 10(-6), and 7.8 x 10(-6) nM(-1) min(-1), respectively), though the activity was considerably lower than that of EndoV (8 x 10(-3) nM(-1) min(-1)). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NO*/O2 delivery system) or ONOOCO2- using a plasmid nicking assay and by LC-MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2-. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.     

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥10(17)M(-1) at 23°C, pH 7.0).     

Additional moults into 'elongatus' males in laboratory-reared Polydesmus angustus Latzel, 1884 (Diplopoda, Polydesmida, Polydesmidae) - implications for taxonomy

The number of stadia during post-embryonic development is supposed to be fixed in most species of the millipede order Polydesmida. For the first time since 1928, additional moults were observed in two males of Polydesmus angustus Latzel, 1884 reared in the laboratory. These 'elongatus' males sensu Verhoeff reached stadium IX instead of stadium VIII, with addition of a further podous ring (32 pairs of legs). One male had well-developed gonopods at stadium VIII, which regressed at stadium IX; the other had no gonopods at stadium VIII, which developed at stadium IX. The two cases correspond to the 'regressionis' and 'progressionis' forms described by Verhoeff in Polydesmus complanatus (Linnaeus, 1761), which confirms entirely his results. Additional moults appear to be associated with small body sizes and possible underlying mechanisms are discussed. Comparisons between millipede orders indicate that post-embryonic development is less strictly canalized in Polydesmida than in Chordeumatida. This implies that the adult number of body rings is of limited taxonomic value in Polydesmida and should not be viewed as a character of generic importance.     

High swimming and metabolic activity in the deep-sea eel Synaphobranchus kaupii revealed by integrated in situ and in vitro measurements

Several complementary studies were undertaken on a single species of deep-sea fish (the eel Synaphobranchus kaupii) within a small temporal and spatial range. In situ experiments on swimming and foraging behaviour, muscle performance, and metabolic rate were performed in the Porcupine Seabight, northeast Atlantic, alongside measurements of temperature and current regime. Deep-water trawling was used to collect eels for studies of animal distribution and for anatomical and biochemical analyses, including white muscle citrate synthase (CS), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and pyruvate kinase (PK) activities. Synaphobranchus kaupii demonstrated whole-animal swimming speeds similar to those of other active deep-sea fish such as Antimora rostrata. Metabolic rates were an order of magnitude higher (31.6 mL kg(-1) h(-1)) than those recorded in other deep-sea scavenging fish. Activities of CS, LDH, MDH, and PK were higher than expected, and all scaled negatively with body mass, indicating a general decrease in muscle energy supply with fish growth. Despite this apparent constraint, observed in situ burst or routine swimming performances scaled in a similar fashion to other studied species. The higher-than-expected metabolic rates and activity levels, and the unusual scaling relationships of both aerobic and anaerobic metabolism enzymes in white muscle, probably reflect the changes in habitat and feeding ecology experienced during ontogeny in this bathyal species.     

Short-timescale variability of picophytoplankton abundance and cellular parameters in surface waters of the Alboran Sea (western Mediterranean)

The Alboran Sea (western Mediterranean) is characterized bya well-defined hydrological structure, the Almeria (Spain)–Oran(Algeria) geostrophic front. During the Almofront-2 cruise (November22, 1997 to January 18, 1998), high frequency sampling (     

The past, present and future of childhood malaria mortality in Africa

During the past few years, there has been a historic series of declarations of renewed commitment to malaria control in Africa. Whether the burden of malaria is increasing in Africa is a moot point. This article attempts to re-construct the evidence for the trends in childhood mortality as a result of Plasmodium falciparum infection over the last century in Africa.     

Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers

Although theskeletal muscle sodium channel is a good substrate for cAMP-dependentprotein kinase (PKA), no functional consequence was observed for thischannel expressed in heterologous systems. Therefore, we investigatedthe effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclicmonophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on thenative sodium channels of freshly dissociated rat skeletal musclefibers by means of the cell-attached patch-clamp technique. Externallyapplied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by~75% with no change in kinetics. Single-channel conductance andnormalized activation curves were unchanged by CPT-cAMP. In contrast,steady-state inactivation curves showed a reduction of the maximalavailable current and a negative shift of the half-inactivationpotential. Similar effects were observed with dibutyryl adenosine3',5'-cyclic monophosphate but not with cAMP, which doesnot easily permeate the cell membrane. Incubation of fibers for 1 hwith 10 µM H-89, a PKA inhibitor, did not prevent the effect ofCPT-cAMP. Finally, the -adrenoreceptor agonist isoproterenolmimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM.These results indicate that cAMP inhibits native skeletal muscle sodiumchannels by acting within the fiber, independently of PKA activation.

     

Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC

In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.